Method 1615

Transcription

Method 1615

EPA/600/R-10/181 | December 2010 | www.epa.gov/ord
Method 1615
Measurement of Enterovirus and Norovirus
Occurrence in Water by Culture and RT-qPCR
Office of Research and Development
National Exposure Research Laboratory, Cincinnati, OH
METHOD 1615. Enterovirus and Norovirus occurrence in water
Cover:
Top picture: Prairie Du Sac, WI Pump house, courtesy of Dr. Mark Borchardt
Bottom left picture: norovirus, courtesy of Fred P. Williams; Bar = 50 nanometers
Bottom right picture: poliovirus, courtesy of Fred P. Williams; Bar = 50 nanometers
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METHOD 1615
MEASUREMENT OF ENTEROVIRUS AND NOROVIRUS
OCCURRENCE IN WATER BY CULTURE AND RT-qPCR
Version 1.0
December 2010
AUTHORS
G. S. Fout,1 N. E. Brinkman,1* J. L. Cashdollar,1* S. M. Griffin,1* B. R. McMinn,1* E. R.
Rhodes,1* E.A. Varughese,1* M. R. Karim,1 A. G. Grimm,1 S. K. Spencer,2 and M. A. Borchardt2
U.S. Environmental Protection Agency, Office of Research and Development,
National Exposure Research Laboratory, Cincinnati, OH;1 and USDA-Agricultural
Research Service,2 Marshfield, WI
*NEB, JLC, SMG, BRM, ERR, and EAV contributed equally to the molecular method
development
DISCLAIMER
This method has been reviewed by the U.S. Environmental Protection Agency (EPA)’s Office of
Research and Development (ORD) and approved for publication. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
ACKNOWLEDGEMENTS
The authors thank Drs. Sandhya Parshionikar, Keya Sen, and Carrie Miller from EPA’s
Office of Water, and Drs. George Di Giovanni from the Texas AgriLife Research Center, El
Paso, TX; Paul Hazelton from the University of Manitoba, Winnipeg, Manitoba, Timothy Straub
from the Pacific Northwest National Laboratory, Richland, WA, and Susan Boutros from
Analytical Associates, Ithaca, NY for reviewing this manuscript and for providing helpful
comments. Mohammad R. Karim was supported through an appointment to the Research
Participation Program at ORD’s National Exposure Research Laboratory, which was
administered by the Oak Ridge Institute for Science and Education through an Interagency
Agreement between the U.S. Department of Energy and EPA. The authors thank Dr. Skip
Virgin of Washington University School of Medicine, St. Louis, MO for Murine norovirus.
Questions regarding this method or its application should be addressed to:
G. Shay Fout, Ph.D.
U.S. Environmental Protection Agency
National Exposure Research Laboratory
Microbiological and Chemical Exposure
Assessment Research Division
Cincinnati, OH 45268-1320
(513) 569-7387
(513) 569-7117 (fax)
[email protected]
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ABBREVIATIONS USED
BGM
BSA
Cat. No.
cDNA
CL
Ct
Cp
Cq
CPE
CV
D
dH2O
DNA
EPA
EV
FCSV
GC
HGV
ICR
LIMS
MEM
MPN
MSDS
Negative FCSV
NoV GI
NoV GII
NPT
NTU
ORD
OSHA
OW
PBS
PCR
PE
QC
qPCR
RT-PCR
RT-qPCR
RNA
RPM
Buffalo Green monkey kidney cells
Bovine serum albumin
Catalog number
Complementary DNA
Confidence limit
Cycle threshold
Crossing point
Quantitation point
Cytopathic effect
Coefficient of variation
Volume of original water sample assayed
Distilled or deionized water
Deoxyribonucleic acid
United States Environmental Protection Agency
Enteroviruses belonging to the genus, Enterovirus
Final concentrated sample volume
Genome copy
Hepatitis G virus
Information Collection Rule
Laboratory information management system
Minimal essential medium
Most probable number
Material Safety Data Sheet
Final concentrated sample volume from a negative QC sample
Genogroup I noroviruses belonging to the genus, Norovirus
Genogroup II noroviruses belonging to the genus, Norovirus
National pipe thread
Nephelometric Turbidity Units
Occupational Safety and Health Administration
Office of Water
Phosphate buffered saline
Polymerase chain reaction
Performance evaluation
Quality control
Quantitative polymerase chain reaction
Reverse transcription-polymerase chain reaction
Reverse transcription-quantitative polymerase chain reaction
Ribonucleic acid
Revolutions per minute
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RT
S
SOP
TCVA
TSV
U.S.
Reverse transcription
Assay sample volume
Standard operating procedure
Total cultural virus assay
Total sample volume
United States
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TABLE OF CONTENTS
ABBREVIATIONS USED ................................................................................................ iii
SCOPE AND APPLICATION ............................................................................................1
BACKGROUND ...........................................................................................................1
METHOD CONSTRAINTS ..........................................................................................3
SUMMARY OF METHOD .................................................................................................3
DEFINITIONS.....................................................................................................................3
INTERFERENCES ..............................................................................................................6
SAFETY ..............................................................................................................................7
EQUIPMENT AND SUPPLIES ..........................................................................................8
REAGENTS, MEDIA, AND STANDARDS ....................................................................12
QUALITY ASSURANCE .................................................................................................16
SAMPLE COLLECTION, PRESERVATION, AND STORAGE....................................20
SAMPLE COLLECTION............................................................................................20
SHIPMENT OF SAMPLES ........................................................................................23
LABORATORY HOLDING TIME AND TEMPERATURE ....................................24
FILTER ELUTION PROCEDURE ...................................................................................24
ELUTION EQUIPMENT SETUP ...............................................................................24
ELUTION ....................................................................................................................25
ORGANIC FLOCCULATION CONCENTRATION PROCEDURE ..............................26
ORGANIC FLOCCULATION....................................................................................26
RECONCENTRATED ELUATE ................................................................................26
TOTAL CULTURABLE VIRUS ASSAY ........................................................................29
QUANTAL ASSAY ....................................................................................................29
VIRUS QUANTITATION ..........................................................................................33
ENTEROVIRUS AND NOROVIRUS MOLECULAR ASSAY ......................................34
PRELIMINARY PROCEDURES ...............................................................................35
TERTIARY CONCENTRATION ...............................................................................35
NUCLEIC ACID ISOLATION ...................................................................................36
REVERSE TRANSCRIPTION (RT) ..........................................................................37
REAL-TIME QUANTITATIVE PCR (qPCR) ...........................................................38
METHOD PERFORMANCE ............................................................................................42
CULTURABLE ASSAY .............................................................................................42
MOLECULAR PROCEDURE ....................................................................................43
STERILIZATION AND DISINFECTION........................................................................44
TABLES AND FIGURES .................................................................................................47
DATA SHEETS .................................................................................................................56
REFERENCES ..................................................................................................................62
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LIST OF TABLES
Table 1. Viruses Detected by EPA Method 1615 ..............................................................47 Table 2. Specified and Recommended Sample Volumes ..................................................47 Table 3. MPN Program Default Settings for MPN Calculator ..........................................48 Table 4. Primers and TaqMan® Probes for Virus Detection by RT-qPCR ........................48 Table 5. RT Master Mix 1 and 2 ........................................................................................49 Table 6. PCR Master Mix for Enterovirus Assay ..............................................................50 Table 7. PCR Master Mix for Norovirus GIA Assay ........................................................50 Table 8. PCR Master Mix for Norovirus GIB Assay.........................................................51 Table 9. PCR Master Mix for Norovirus GII Assay ..........................................................51 Table 10. PCR Master Mix for Hepatitis G Assay ............................................................52 Table 11. Mean Recovery and Coefficient of Variation Range.........................................52 LIST OF FIGURES
Figure 1. Uninfected BGM Cells .......................................................................................53 Figure 2. Early CPE from Poliovirus .................................................................................53 Figure 3. Sample Filtration Apparatus ...............................................................................54 Figure 4. Elution of an Electropositive Filter with Beef Extract .......................................54 Figure 5. RT-qPCR Schematic ..........................................................................................55 vi
1.
SCOPE AND APPLICATION
1.1.
BACKGROUND
1.1.1.
Introduction
1.1.1.1.
EPA Method 1615 provides culture and molecular procedures
for detecting human enteroviruses, human noroviruses, and
mammalian orthoreoviruses (culture procedure only) in water
(Table 1). The cell culture procedure detects enterovirus and
orthoreovirus species that are capable of infecting and
producing cytopathic effects (CPE) in the Buffalo Green
monkey kidney (BGM) cell line (16, 19). Although this cell
line is considered a “gold standard” for detection of infectious
waterborne viruses, noroviruses and a number of enteroviruses
do not replicate in BGM cells. There is no established cell line
for detection of infectious human noroviruses, but a prototype
research method is under development (37, 38). The molecular
procedure incorporated into EPA Method 1615 detects the
noroviruses and enteroviruses shown in Table 1, including
those enteroviruses that do not replicate on BGM cells.
However, it should be noted that a positive result indicates the
presence of viral nucleic acid and not the infectivity status of
the detected virus.
1.1.1.2.
Enteroviruses and noroviruses are enteric viruses that replicate
within the gastrointestinal tract and are spread through the
fecal-oral route. They cause a variety of waterborne infections
through exposure to contaminated drinking and recreational
waters. Infections may be asymptomatic or result in mild
gastroenteritis, febrile illness, or respiratory symptoms. They
can also cause a variety of serious diseases such as aseptic
meningitis, encephalitis, flaccid paralysis, hand, foot and
mouth disease, hemorrhagic conjunctivitis, myocarditis,
neonatal sepsis-like disease, or severe gastroenteritis (3, 20,
25). Enteroviruses and noroviruses have not only been found
in drinking and recreational waters, but have caused
waterborne outbreaks (4, 5, 18, 26, 27, 41). Due to public
health concern, these viruses are on EPA’s Contaminant
Candidate List 3 (http://www.epa.gov/safewater/ccl/ccl3.html).
The Mammalian orthoreovirus species is not associated with
any known waterborne outbreaks and usually does not cause
disease in humans (13, 15). If desired, orthoreoviruses can be
assayed using the molecular method found in Fout et al. (18).
1.1.1.3.
Molecular procedures, such as polymerase chain reaction
(PCR) and reverse transcription-PCR (RT-PCR), provide the
flexibility to detect all waterborne human enteric viruses for
which genome sequence data is available (18). The advent of
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real time quantitative PCR (qPCR) has resulted in additional
advantages over conventional PCR in that quantitative results
can be obtained in a very short time (21). These molecular
methods have been widely used to detect viruses in
environmental waters (7, 8, 22, 27, 28, 33, 39). Despite the
advantages, molecular techniques are subject to three main
limitations. First, PCR methods assay smaller volumes than
culture methods, resulting in lower detection limits. Second,
these methods are sensitive to inhibitors that are present in
some environmental samples. To address this problem,
controls are used to determine whether negative results are true
negative or false negative values. Finally, molecular methods
do not distinguish between infectious and noninfectious
viruses. Therefore, a positive PCR assay for a particular
pathogen in drinking water does not directly address issues of
public health. Research is ongoing on several promising
approaches to detect infectious viruses (31, 34). However,
PCR is still a useful public health tool in spite of these
problems. Because there is a strong relationship between
indicator measurements by qPCR and health effects in
recreational waters (40), EPA is considering using qPCR to set
new criteria for monitoring recreational beaches. At the very
least, positive PCR virus findings provide a warning of
possible contamination issues, but recent studies have indicated
a direct relationship between health effects and positive RTqPCR findings for human viruses (Borchardt et al., manuscript
in preparation).
1.1.2.
Development of the ICR Total Culturable Virus Assay - In the 1990s EPA
issued an Information Collection Rule (ICR; Federal Register 61:2435324388) that required all drinking water utilities serving a population over
100,000 to monitor their source water for viruses monthly for a period of
18 months. The Rule also required that systems finding greater than one
infectious enteric virus particle per liter of source water to monitor their
finished water on a monthly basis. One of the purposes of the Rule was to
obtain national data on virus levels in source waters to determine the
adequacy of treatment requirements. To support the Rule, a virus
monitoring protocol was developed by virologists at the EPA and
modified to reflect consensus agreements from the scientific community
and public comments to the draft rule (19). This standard ICR Total
Culturable Virus method, along with quality assurance and laboratory
approval procedures (http://www.epa.gov/microbes/icrmicro.pdf), was
incorporated into the ICR by reference. The results of the ICR survey
indicated that culturable viruses were present in 24% of the source waters
throughout the nation. Since the end of the ICR, the ICR Total Culturable
Virus method has continued to be used in the U.S. and in international
settings for the detection of culturable viruses in surface, ground, and
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treated waters (14, 19, 24), but the high cost of collecting and analyzing
virus samples has limited the method’s widespread use.
1.1.3.
1.2.
2.
Development of Method 1615 – In the past few years, an alternative
sampling protocol that significantly reduces the cost of sampling has been
found to be equivalent in performance to the ICR method (24). Method
1615 is a modification of the ICR protocol. It incorporates the alternative
sampling procedure and reduces the number of required cell culture
replicates required by the ICR protocol. It also includes a molecular
procedure that is a modification of a method used to survey groundwaters
for enteric viruses in Wisconsin (9, 27).
METHOD CONSTRAINTS
1.2.1.
This method is for use by analysts skilled in virus concentration, elution,
cell culture, and molecular techniques.
1.2.2.
Analysts must not deviate from any of the procedures described in this
method if the data is being generated to fulfill EPA regulatory
requirements. For example, alternative procedures for elution, secondary
and tertiary concentration, and analyses by culture and RT-qPCR must not
be used without prior approval by EPA.
SUMMARY OF METHOD
Viruses that may be present in environmental or finished drinking waters are concentrated
by passage through electropositive filters. Viruses are eluted from the filters with a beef
extract reagent and concentrated using organic flocculation. A portion of the concentrated
eluate is then inoculated onto replicate flasks of BGM cells to measure infectious viruses.
Cultures are examined for the development of cytopathic effects for two weeks and then repassaged onto fresh cultures for confirmation. Virus concentration in each sample is
calculated in terms of the most probable number (MPN) of infectious units per liter using
EPA’s MPN calculator. For molecular assays, the concentrated eluate is concentrated
again by centrifugal ultrafiltration. The RNA is extracted from the concentrate and tested
for enterovirus and norovirus RNA using real time quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR). Virus concentrations for the molecular assay are
calculated in terms of genomic copies of viral RNA per liter based upon a standard curve.
3.
DEFINITIONS
3.1.
ANALYSIS BATCH
3.1.1.
All virus samples processed by an analyst within one week's span shall be
considered a "batch". A week is defined as a 7-day period.
3.1.2.
Each sample result must be associated with a batch number.
3.1.3.
A positive and negative Quality Control (QC) sample set must be
processed with each batch.
3.1.3.1.
Failure to obtain both a positive value in the positive QC
sample and a negative value in the negative QC sample results
in failure of the entire batch.
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3.1.3.2.
3.2.
Obtaining a positive value in the positive QC sample and a
negative value in the negative QC sample results in acceptance
of the data from the entire batch.
BUFFALO GREEN MONKEY KIDNEY (BGM) CELLS
This is a stable cell line of monkey kidney cells that were originally developed at
the University of Buffalo for clinical isolation of enteroviruses and later adapted for
use in detecting infectious viruses in environmental samples (16). BGM cells form
a monolayer of cells when propagated in tissue culture vessels. Figure 1 is a
micrograph of uninfected BGM cells growing as a monolayer.
3.3.
CONTAMINANT CANDIDATE LIST (CCL)
A list of chemicals and microbial agents under consideration for regulatory action
by EPA. The current list may be obtained from
http://water.epa.gov/scitech/drinkingwater/dws/ccl/.
3.4.
CYTOPATHIC EFFECT (CPE)
The degeneration of cells caused by virus replication. It often involves the
complete disintegration of cells. It may also be identified through changes in cell
morphology. However, care must be taken in the use of changes in cell
morphology as evidence of CPE, because uninfected BGM cells change
morphology during mitosis. True CPE is always progressive and can be rated on a
0-4 scale, with the values 0, 1, 2, 3, and 4 indicating that 0% (Figure 1), 25%
(Figure 2), 50%, 75%, and 100% of the monolayer is showing CPE, respectively.
Additional examples of CPE can be found in Malherbe and Strickland-Cholmley
(29).
3.5.
CYTOTOXICITY
Cytotoxicity is the development of CPE from toxic components in the matrix.
Cytotoxicity can be distinguished from viral CPE by its early development after
sample inoculation or by the failure to observe CPE in the second passage required
by this method. Unlike CPE, which begins as small clusters of killed cells (see
Figure 2) after two or more days of incubation, cytotoxicity usually develops
uniformly in all inoculated cell culture replicates within 24 hours of inoculation.
3.6.
DETECTION LIMIT
The number of virus particles or genome copy numbers that can be detected in a
given volume by a method with 95% confidence.
3.7.
ENTERIC VIRUSES
Viruses that primarily infect and replicate in the gastrointestinal tract are known as
enteric viruses. These include enteroviruses, noroviruses, rotaviruses, hepatitis A
virus, adenoviruses, and reoviruses, among others. Enteric viruses are capable of
causing a wide range of illnesses such as gastroenteritis, paralysis, aseptic
meningitis, respiratory illness, fevers, myocarditis, and neonatal enteroviral sepsis.
Enteric viruses can be present in human and animal feces, which can contaminate
recreational and drinking water sources.
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3.8.
ENTEROVIRUS
Enteroviruses are a genus in the Picornaviridae family. These viruses are among
the most common viruses infecting humans worldwide. Enteroviruses are small
(approximately 30 nm), nonenveloped, single-stranded, positive sense RNA viruses
with an icosahedral capsid. Traditionally, human enterovirus serotypes have been
classified into echoviruses, coxsackieviruses group A and B, and polioviruses.
Current taxonomy based on molecular typing divides human enteroviruses into four
species, Human enterovirus A, B, C, and D.
3.9.
GROWTH AND MAINTENANCE MEDIUM
Growth medium consisting of a 50/50 (v/v) mixture of Minimum Essential Medium
(MEM) and Leibovitz's L-15 medium with antibiotics and 10% bovine serum is
recommended. Maintenance medium consists of a 50/50 mixture of Eagle's
minimum essential medium (MEM) and Leibovitz's L-15 medium with antibiotics
and 2% bovine serum.
3.10. INOCULATION
Inoculation is the placement of concentrated water samples onto a monolayer of
cells in a culture vessel for growing or replication of viruses in the cells.
3.11. MATERIAL SAFETY DATA SHEETS (MSDS)
These sheets contain written information provided by vendors concerning a
chemical’s toxicity, health hazards, physical properties, fire, and reactivity data,
including storage, spill, and handling precautions.
3.12. MONOLAYER
A single confluent layer of cells covering the bottom of a tissue culture dish or flask
(Figure 1).
3.13. NOROVIRUS
Noroviruses constitute a genus in the Caliciviridae family. The genus is divided
into five genogroups (GI-GV) and 29 genetic clusters (42). Noroviruses are
recognized as a leading cause of nonbacterial gastroenteritis in humans.
Noroviruses are small (approximately 27 nm) and the genome possess a positive
sense, single stranded RNA in a nonenveloped icosahedral capsid. Due to the
absence of a standardized and validated infectivity assay for human noroviruses, the
presence of noroviruses in environmental waters must be measured using molecular
methods.
3.14. PERFORMANCE EVALUATION/PERFORMANCE TEST SAMPLE (PE)
A sample containing Sabin poliovirus type 3 at a concentration unknown to the
analysts. PE samples are typically sent to all laboratories performing the method
from a single outside source when performance evaluation is required by EPA. The
purpose of the PE sample is to demonstrate that all analysts performing the method
can meet minimum performance standards.
3.15. QUALITY CONTROL SAMPLE (QC)
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This is a sample containing Sabin poliovirus type 3 at a known concentration. QC
samples will typically be sent to all laboratories performing the method from a
single outside source when performance evaluation is required by EPA. The
purposes of the QC sample are to give laboratories a standard sample for training
new analysts and to give EPA and laboratory QA officials a tool to evaluate method
performance for all laboratory analysts.
3.16. QUANTITATIVE CYCLE [(Cq), also called Cycle Threshold (Ct) or Crossing
Point (Cp)]
The cycle at which the fluorescence of a quantitative PCR assay crosses the
threshold that defines a positive reaction or at which the second derivative
maximum is reached (10, 11).
3.17. QUANTITATIVE POLYMERASE CHAIN REACTION (qPCR) and
REVERSE TRANSCRIPTION-qPCR (RT-qPCR)
This is a procedure for quantitatively detecting the levels of specific DNA in a
sample, or following reverse transcription [RT, e.g., the synthesis of complementary
DNA (cDNA) from RNA], the levels of specific RNA (e.g., viral) in a sample.
3.18. REAGENT WATER
This is deionized or distilled reagent grade water (dH2O) with a resistivity greater
than 1 Siemens per meter (S/m), i.e., 1 megohms-cm at 25ºC; if available, reagent
grade water with a resistivity greater than 0.1 S/m (10 megohms-cm) is preferred
(2).
3.19. STANDARD OPERATING PROCEDURE
A Standard Operating Procedure (SOP) is a set of written instructions that document a
routine or repetitive activity followed by an organization. The development and use of
SOPs are an integral part of a successful quality system as it provides individuals with
the information to perform a job properly, and facilitates consistency in the quality and
integrity of data. EPA guidance on developing SOPs can be obtained from
www.epa.gov/quality/qs-docs/g6-final.pdf.
4.
INTERFERENCES
4.1.
REAGENTS
To minimize cross contamination, Analytical Reagent or ACS grade chemicals
(unless specified otherwise) and reagent water should be used to prepare all media
and reagents. It is recommended that water, media, and other reagent solutions be
purchased from commercial sources and that tissue culture grade water be used for
preparation of tissue culture media.
4.2.
MATRIX INTERFERENCE
Matrix interferences may lead to false negative results and are caused by colloidal,
suspended, or dissolved substances that are present in the water sample. Matrix
interference can vary across different water sources and even across time in the
same source.
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4.3.
5.
4.2.1.
Matrix interference due to colloidal or suspended solids may reduce the
water volume that can be passed through the positively charged filters
used in this method. Prefilters (item 6.1.5) or more than one
electropositive filter must be used to overcome this type of interference.
4.2.2.
Matrix interference may be identified by its effects on the culture or
molecular assays. This may be expressed as the development of
cytotoxicity in culture assays and or by inhibition of molecular assays.
OTHER INTERFERENCE
4.3.1.
Failure to dechlorinate treated tap water samples during sampling or
prolonged exposure to ambient temperatures during sample transportation
or in the laboratory can lead to virus loss.
4.3.2.
Inadequate disinfection of the sampling apparatus and contamination of
reagents and supplies can lead to sample contamination. Inadequate
disinfection of the sampling apparatus is identified using negative Quality
Control samples (Section 8.4.3).
4.3.3.
Inadequate physical separation and controlled workflow may lead to PCR
interference due to false positive results from contamination. EPA’s
guidance for processing and handling these samples must be followed to
minimize this interference (36).
SAFETY
5.1.
SAFETY PLAN
The biohazard associated with, and the risk of infection from, human enteric viruses
is high in this method because potentially infectious viruses are handled. This
method does not purport to address all the safety issues associated with its use.
Each laboratory is responsible for establishing a safety plan that addresses
appropriate safety and health practices prior to using this method. In particular,
laboratory staff must know and observe the safety procedures required in a
microbiology laboratory that handles pathogenic organisms while preparing, using,
and disposing of sample concentrates, reagents and materials, and while operating
sterilization equipment. Minimum requirements have been published by the U.S.
Department of Health and Human Services (1).
5.2.
SHIPMENT OF SAMPLES
The samples collected using this method may be shipped as non-infectious
materials, unless they are known to contain virus or other infectious materials. If
they are known to contain infectious materials, laboratories are responsible for
packaging and shipping them according to all Department of Transportation,
Centers for Disease Control and Prevention, and State regulations.
5.3.
CHEMICAL SAFETY
Each laboratory is responsible for the safe handling of the chemicals used in this
method. The OSHA laboratory standards can be found on line at
http://www.osha.gov/SLTC/laboratories/standards.html.
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6.
EQUIPMENT AND SUPPLIES
References to specific brands or catalog numbers are included as examples only and do not
imply endorsement of the product. These references do not preclude the use of other
vendors or supplies. However, equivalent method performance should be demonstrated for
substitutions of the electropositive filter, beef extract, and qPCR reagents. All equipment
should be cleaned according to the manufacturers’ recommendations. Disposable supplies
should be used wherever possible to reduce the possibility of cross contamination. Note:
Storage of samples and reagents at temperatures below 0°C must be done using manual
defrost freezers.
6.1.
SAMPLE FILTRATION APPARATUS
Figure 3 shows the Sample Filtration Apparatus which has been modified from that
given in Fout et al. (19) for use with the NanoCeram® electropositive cartridge
filter. The modification also increases the efficacy for disinfecting the apparatus.
The sample filtration apparatus shown in Figure 1 is recommended, but that shown
in Figure VIII-1 of Fout et al. (19) also may be used for virus monitoring. The
apparatus may have to be modified using appropriate size housings and adapters for
use with other equivalent filters, such as the ten-inch 1MDS Virosorb cartridge
filter. The current configuration does not use a pressure regulator or pressure
gauge, as these components are difficult to disinfect and subject to corrosion;
however, laboratories are responsible for ensuring that water pressure at sampling
sites does not exceed the pressure ratings of the cartridge housings used (125 psi for
item 6.1.2.2).
6.1.1.
6.1.2.
6.1.3.
Intake Module
6.1.1.1.
Backflow regulator (Watts Regulator Series 8 C Hose
Connection Vacuum Breaker; this component is optional)
6.1.1.2.
Swivel female insert with garden hose threads (United States
Plastic Cat. No. 63003)
6.1.1.3.
½” tubing (Cole-Parmer Cat. No. 06601-03) and hose clamps
(Cole-Parmer Cat. No. 06403-10)
6.1.1.4.
½” hose barb quick disconnect body (Cole Parmer Cat. No.
31307-11)
6.1.1.5.
Thread tape (Cole Parmer Cat. No. 08270-34)
Cartridge Housing Module for NanoCeram filters
6.1.2.1.
½” NPT (M) quick disconnect insert (Cole Parmer Cat. No.
31307-31; connected to the inlet port of the cartridge housing)
6.1.2.2.
Cartridge housing (Argonide Cat. No. H2.5-5)
6.1.2.3.
½” NPT (M) quick disconnect body (Cole Parmer Cat. No.
31307-06; connected to the outlet port of the cartridge housing)
6.1.2.4.
5” NanoCeram cartridge filter (Argonide Cat. No. VS2.5-5).
Discharge Module
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6.1.4.
6.1.3.1.
½” NPT (M) quick disconnect insert (Cole Parmer Cat. No.
31307-31)
6.1.3.2.
½” NPT (F) straight connector (Cole Parmer Cat. No. 0634903)
6.1.3.3.
Flow meter (Flow Technology Cat. No. FT6-8NENWULEG-3)
– Note: the flow meter calibration at the flow rates used for
sampling should be confirmed before use and after every
month of use. The calibration can be performed by measuring
the time to fill a 4 L or larger graduated cylinder (Cole Parmer
Cat. No. 06135-90; e.g., 24± 1 second for the 10 liter per
minute rate).
6.1.3.4.
Rate/Totalizer (Flow Technology Cat. No. BR30-5-A-4)
6.1.3.5.
½” NPT (F) straight connector (Cole Parmer Cat. No. 0634903)
6.1.3.6.
¾” NPT (M) × ½” NPT (M) reduction nipple (Cole Parmer
Cat. No. 06349-87)
6.1.3.7.
¾” NPT (F) bronze globe valve (Cole Parmer Cat. No. 9867509)
6.1.3.8.
¾” NPT (M) × GHT(M) fitting (United States Plastic Cat. No.
63016)
6.1.3.9.
A garden hose of sufficient length to reach a drain.
Injector module – this module, prepared using the components below,
should only be used when it is necessary to add thiosulfate or other
additives to water during sampling. It can be modified into a double
injector module for the addition of more than one additive by installing a
small ¼” NPT (F) TEE (Cole Parmer Cat. No. 06349-51) on top of the
larger TEE (item 6.1.4.2) via the male reducer (item 6.1.4.4) and placing
items 6.1.4.5 and 6.1.4.6 on each end of the smaller TEE using a ¼” NPT
(M) Nipple (Cole Parmer Cat No. 30623-25).
6.1.4.1.
⅜” NPT (M) quick disconnect insert (Cole Parmer Cat. No.
31307-30, attached to the left port of the TEE)
6.1.4.2.
⅜” NPT (F) TEE (Cole Parmer Cat. No. 06482-84)
6.1.4.3.
⅜” NPT (M) quick disconnect body (Cole Parmer Cat. No.
31307-05, attached to the right port of the TEE)
6.1.4.4.
⅜” NPT (M) × ¼” NPT (M) male reducer (Cole Parmer Cat.
No. 30623-42; this and following components are connected to
the top port of the TEE)
6.1.4.5.
¼” NPT (F) metallic check valve (CV; Cole Parmer Cat. No.
98676-00)
9
6.1.5.
6.1.6.
6.2.
6.1.4.6.
¼” NPT (M) × ¼” tubing ID male pipe adaptor elbow (Cole
Parmer Cat. No. 30622-97)
6.1.4.7.
15-gallon chemical tank (Grainger Cat. No. 4UP09) with
tubing. This item is for injecting 2% thiosulfate (item 7.3) into
water containing a disinfectant. The container size can be
adjusted to meet the anticipated need.
6.1.4.8.
Chemical metering pump (Grainger Cat. No. 4UP03; before
first use, adjust the chemical metering pump to deliver 2.4 or 6
mL/minute (i.e., 0.6 mL × L of disinfected water passing
through the sample filtration apparatus each minute) for flow
rates of 4 or 10 L/minute, respectively. Using a graduated
cylinder to measure the flow rate, and record or mark the pump
setting for each rate.
Prefilter module – for use with waters exceeding 75 nephelometric
turbidity units (NTU) or for other conditions that prevent the minimum
sampling volumes from being obtained. Note: The NanoCeram filter is
more susceptible to clogging than the 1MDS filter. A prefilter module
may be required for some matrices even when the turbidity is considerably
below 75 NTU.
6.1.5.1.
Prepare the prefilter module as described step 6.1.2, except use
the filter described in step 6.1.5.2
6.1.5.2.
10 μm Polypropylene Prefilter Cartridge (Parker Hannifin Cat.
No. M19R5-A)
Assemble the modules as shown in Figure 3 using thread tape (item
6.1.1.5) on all connections. Sterilize the regulator, prefilter housing, and
cartridge housings with chlorine as described in Section 15. Using aseptic
technique, add a sterile NanoCeram or 1MDS cartridge to the cartridge
housing and, if needed, a presterilized polypropylene cartridge to the
prefilter housing. Cover the ends with sterile aluminum foil.
OTHER SAMPLING AND SHIPPING MATERIALS
6.2.1.
Peristaltic or chemical resistant pump capable of pumping water at 4-10
liters/min and appropriate connectors (for use where garden hose-type
pressurized taps for the source or finished water to be monitored are
unavailable and for QC samples). Follow the manufacturer's
recommendations for pump priming.
6.2.2.
One L polypropylene wide-mouth bottles (Nalgene Cat. No. 2104-0032)
6.2.3.
Collapsible 10 L LDPE cubitainer (Cole Parmer Cat. No. 06100-30)
6.2.4.
Portable pH and temperature probe (Omega Cat. No. PHH-830)
6.2.5.
Portable turbidity meter (Omega Cat. No. TRB-2020)
6.2.6.
Portable Chlorine (Free & Total), Pocket Colorimeter II Test Kit with
reagents (Hach Cat. No. 5870062).
10
6.3.
6.2.7.
Commercial ice packs (Cole-Parmer Cat. No. 06345-20)
6.2.8.
iButtons (Maxim Cat. No. DS1921G)
6.2.9.
Insulated shipping box with carrying strap (17" × 17" × 16"; Cole-Parmer
Cat. No. 03742-00 and 03742-30) or insulated storage and transport chest
(Fisher Scientific Cat. No. 39)
6.2.10.
Miscellaneous - aluminum foil, surgical gloves, waterproof marker
EQUIPMENT AND SUPPLIES FOR THE ORGANIC FLOCCULATION
PROCEDURE
6.3.1.
Dispensing pressure vessels, 5 and 20 L capacity (Millipore Corp. Cat.
No. XX6700P05 and XX6700P20)
6.3.2.
Pressure source - laboratory positive pressure air line (equipped with oil
filter), compressed nitrogen, peristaltic pump (e.g., Cole Parmer Cat. No.
07523-60), or self-priming pump (e.g., Cole Parmer Cat. No. 07036-10)
6.3.3.
pH meter with combination-type electrode and an accuracy of at least 0.1
pH units
6.3.4.
Magnetic stirrer and stir bars
6.3.5.
Refrigerator, set at 4ºC ± 3ºC
6.3.6.
Refrigerated centrifuge capable of attaining 4,000 ×g
6.3.6.1.
Screw-capped centrifuge bottles with 100 to 1000 mL capacity.
Each bottle must be rated for the relative centrifugal force
used.
6.3.6.2.
Appropriate centrifuge buckets for centrifuge bottles
6.3.7.
Sterilizing filter – 0.22 μm Acrodisc filter with prefilter (VWR Cat. No.
28143-295) for use at step 11.2.3.8.
6.3.8.
47 mm disc filter holder (Millipore Corp. Product No. SX0004700)
6.3.9.
Sterilizing filter stack – Place a 0.22 μm pore-size membrane filter
(Millipore Corp. Product No. GSWP04700) on the bottom of a 47 mm
disc filter holder.
6.3.9.1.
Place an AP15 prefilter (Millipore Corp. AP1504700) on top of
the 0.22 μm filter and an AP20 prefilter (Millipore Corp.
AP2004700) on top of the AP15 prefilter.
6.3.9.2.
Assemble the filter holder unit and sterilize as defined in
section 15.2.2.
6.3.9.3.
Note: Disassemble the filter stack after each use to check the
integrity of the 0.22 μm filter. Refilter any media filtered with
a damaged stack using another sterile sterilizing filter stack.
6.3.9.4.
Note: The sterilizing filter stack is optional, but should be used
for samples that are difficult to filter using item 6.3.7.
11
6.3.10.
6.4.
6.5.
7.
Sterilizing filter – 0.22 μm (Corning Cat. No. 431219) for use at step
12.1.4.2.
EQUIPMENT AND SUPPLIES FOR THE TOTAL CULTURABLE VIRUS
ASSAY
6.4.1.
Mechanical rocking platform (Daigger Product No. EF4907G)
6.4.2.
Incubator capable of maintaining the temperature of cell cultures at
36.5±1°C
6.4.3.
Flasks or bottles for BGM culture maintenance and quantal assays (e.g.,
2100 cm2 roller bottles, Fisher Cat. No. 73520-420; 850 cm2 roller bottles,
Corning Cat. No. 430849; 25 cm2 flasks, Sigma Cat. No. C6231)
EQUIPMENT FOR THE TERTIARY CONCENTRATION PROCEDURE
AND THE MOLECULAR ASSAYS
6.5.1.
UV-Vis Spectrophotometer (Thermo Fisher Scientific Cat. No. NanoDrop
2000)
6.5.2.
Vivaspin 20 – 30,000 MWCO (Sartorius-Stedim Cat. No. VS2022; other
centrifugal concentrators with 30,000 MWCO may be substituted for this
item, if equivalent recoveries are demonstrated)
6.5.3.
50 mL polypropylene centrifuge tube and appropriate buckets for
centrifuge (item 6.3.6)
6.5.4.
Microcentrifuge capable of 20,000 x g (Fisher Scientific Cat. No. 05-40611)
6.5.5.
1.5 mL microcentrifuge tubes (Fisher Scientific Cat. No. 02-682-550)
6.5.6.
Vortex mixer (Fisher Scientific Cat. No. 02-216-100)
6.5.7.
Thermal cycler (Applied Biosystems Cat. No. 4314879)
6.5.8.
Optical reaction plate (Applied Biosystems Cat. No. 4314320)
6.5.9.
Real-Time PCR system (Applied Biosystems Cat. No. 4351405)
REAGENTS, MEDIA, AND STANDARDS
The amount of reagents, media, and standards prepared for each step of the method may be
adjusted proportionally to the number of samples to be analyzed. For any given section of
this method only media, reagents, and standards that are not described in previous sections
are listed.
7.1.
REAGENTS FOR THE SAMPLING PROCEDURE
7.1.1.
Hydrochloric acid (HCl) – Prepare 0.12, 1.2, and 6 M solutions by mixing
50, 100, or 50 mL of concentrated HCl (Note: HCl at 37% concentration
is about 12 M) with 4950, 900, or 50 mL of dH2O, respectively. Prepare
solutions to be used for adjusting the pH of water samples at least 24 h
before use. HCl solutions can be stored for several months at room
temperature.
12
7.2.
7.1.2.
QC stock – This is a stock of Sabin poliovirus type 3 containing 200
MPN/mL. This stock may be prepared by the analytical laboratory or, if
available, obtained from a contractor designated by the US EPA.
7.1.3.
Matrix spike – This is a stock of Sabin poliovirus type 3 containing 1000
MPN/mL. This stock may be prepared by the analytical laboratory or, if
available, obtained from a contractor designated by the US EPA.
7.1.4.
Sodium hypochlorite (NaClO) – Prepare a 0.525% NaClO solution by
diluting household bleach 1:10 in water. 0.525% NaClO solutions can be
stored for one week at room temperature.
7.1.5.
1 M sodium thiosulfate (Na2S2O3) pentahydrate – Prepare a 1 M solution
by dissolving 248.2 g of Na2S2O3 in 1 liter of dH2O. Sodium thiosulfate
solutions may be stored for six months at room temperature.
7.1.6.
2 % sodium thiosulfate (Na2S2O3) pentahydrate – Prepare 2% thiosulfate
by dissolving 1 kg of sodium thiosulfate pentahydrate into 49 L of sterile
water. Sodium thiosulfate solutions may be stored for six months at room
temperature.
REAGENTS FOR THE ELUTION AND ORGANIC FLOCCULATION
PROCEDURES
7.2.1.
Sodium hydroxide (NaOH) – Prepare 0.1, 1, and 5 M solutions by
dissolving 0.4, 4, or 20 g of NaOH in a final volume of 100 mL of dH2O,
respectively. NaOH solutions may be stored for several months at room
temperature.
7.2.2.
0.5% iodine – Prepare a 0.5% iodine solution by dissolving 5 g of iodine
in 1 L of 70% ethanol. Iodine solutions can be stored for one year at room
temperature.
7.2.3.
1.5% beef extract, pH 9.0
7.2.3.1.
Prepare buffered 1.5% beef extract by dissolving 30 g of beef
extract, desiccated (BD Bacto Cat. No. 211520) powder and
7.5 g of glycine (final glycine concentration = 0.05 M) in 1.9 L
of dH2O.
7.2.3.2.
Adjust the pH to 9.0 with 1 or 5 M NaOH and bring the final
volume to 2 L with dH2O.
7.2.3.3.
Autoclave the beef extract solution at 121°C for 15 minutes
and use at room temperature. Beef extract solutions may be
stored overnight at room temperature or 4°C, for one week at
4°C, or for longer periods at -20°C.
7.2.3.4.
Warning: Desiccated beef extract lots show considerable
variation in virus recovery. Therefore, each new lot of beef
extract must be screened before use in the Organic Flocculation
Concentration Procedure to determine whether virus recoveries
are adequate. Perform the screening by spiking one liter of
13
beef extract solution with 1 mL of a QC sample. Process the
spiked sample according to the Organic Flocculation and Total
Culturable Virus Assay procedures given below. The mean
recovery of poliovirus for three trials should be greater than
50%.
7.3.
7.2.4.
1.5% beef extract, pH 7.0 – 7.5 - Prepare 1.5% beef extract by dissolving
7.5 g of beef extract, desiccated powder and 1.88 g of glycine in 0.5 L of
dH2O. Autoclave the beef extract solution at 121°C for 15 minutes and
use at room temperature. This beef extract solution may be stored for up
to six months at room temperature, but must be discarded if there is
evidence of microbial growth or any other change in appearance.
7.2.5.
0.15 M sodium phosphate, pH 9.0 – Prepare 0.15M sodium phosphate by
dissolving 40.2 g of sodium phosphate, dibasic (Na2HPO4 • 7H2O) in a
final volume of 1000 mL dH2O. Adjust the pH to 9.0 with HCl, if
necessary. Autoclave at 121°C for 15 minutes. Sodium phosphate
solutions (items 7.9 and 7.10) may be stored at room temperature for up to
12 months.
7.2.6.
0.15 M sodium phosphate, pH 7.0-7.5 – Prepare by dissolving 40.2 g of
sodium phosphate, dibasic (Na2HPO4 • 7H2O) in a final volume of 1000
mL dH2O. Adjust the pH to 7.0 to 7.5 with HCl. Autoclave at 121°C for
15 minutes.
7.2.7.
Antifoam (Sigma Cat. No. A8311)
REAGENTS FOR THE TOTAL CULTURABLE VIRUS ASSAY
7.3.1.
Cell culture test vessels containing confluent monolayers of BGM cells –
BGM cells between passages 117 and 250 must be used. Cells should be
passaged and maintained using the standard procedures available in the
most recent version of the USEPA Manual of Methods for Virology (6),
available at http://www.epa.gov/microbes/about.html. BGM cells from
various sources and other standard tissue culture techniques and media
may be used as long as analysts meet the acceptance criteria listed in
section 14. Cell cultures used for virus assay are generally found to be at
their most sensitive level between the third and sixth days after their most
recent passage; those older than seven days must not be used.
7.3.2.
Cell culture media
7.3.2.1.
Hank’s balanced salt solution (Invitrogen Cat. No. 14170-112)
7.3.2.2.
Fetal bovine serum, certified, heat-inactivated (Invitrogen Cat.
No. 10082-139)
7.3.2.3.
Trypsin with EDTA (Invitrogen Cat. No. 25300-062)
7.3.2.4.
Fungizone (Invitrogen Cat. No. 15290-018), PenicillinStreptomycin (Invitrogen Cat. No. 15140-122)
14
7.3.3.
7.4.
7.3.2.5.
Minimum essential medium (MEM) with Hanks' salts and Lglutamine (Sigma Aldrich Cat. No. M1018)
7.3.2.6.
Leibovitz's L-15 medium with L-glutamine (Sigma Aldrich
Cat. No. L4386).
7.3.2.7.
7.5% Sodium bicarbonate (Sigma Aldrich Cat. No. S8761)
7.3.2.8.
Trypan blue (Sigma-Aldrich Cat. No. T8154)
Positive Assay Control – Prepare by diluting the QC stock in sodium
phosphate, pH 7.0 - 7.5, to give a concentration of 20 PFU per Inoculation
Volume (see step 11.2.4.5 for a definition of Inoculum Volume).
REAGENTS FOR THE TERTIARY CONCENTRATION PROCEDURE
AND THE MOLECULAR ASSAYS
7.4.1.
Dulbecco’s Phosphate Buffered Saline, without CaCl2 and MgCl2 (U.S
Biological Cat. No. D9820)
7.4.2.
BSA, crystalline (United States Biochemical Cat. No. 10856)
7.4.3.
0.2 μm sterilizing filter (Sigma Cat. No. F-9768)
7.4.4.
5% BSA – Prepare 5% BSA by dissolving 5 g of in 100 mL of dH2O.
Sterilize by passing the solution through a sterilizing filter (item 7.4.3).
Store at 4°C.
7.4.5.
PBS, 0.2% BSA – Prepare by adding 4 mL of 5% BSA to 96 mL of PBS.
Sterilize by passing the solution through a 0.2 μm sterilizing. Store at
4°C.
7.4.6.
QIAamp DNA Blood Mini Kit (Qiagen Cat. No. 51104 or 51106)
7.4.7.
Buffer AVL (Qiagen Cat. No. 19073; carrier RNA is supplied with this
reagent)
7.4.8.
Buffer AVE (Qiagen Cat. No. 1026956)
7.4.9.
Collection tubes, 2 mL (Qiagen Cat. No. 19201
7.4.10.
Absolute ethanol (Fisher Scientific Cat. No. BP2818-100)
7.4.11.
Random Primers, 0.5 μg/μL (Promega Cat. No. C1181)
7.4.12.
10X PCR Buffer II with 25 mM MgCl2 (Applied Biosystems Cat. No.
N808130)
7.4.13.
Deoxyribonucleotides, 10 mM (NTPs; Promega Cat. No. C1141)
7.4.14.
Dithiothreitol, 100 mM (DTT; Promega Cat. No. P1171)
7.4.15.
RNasin® Plus RNase Inhibitor, 40 units/μL (Promega Cat. No. N2615)
7.4.16.
SuperScript II Reverse Transcriptase, 200 units/μL (Invitrogen Cat. No.
18064)
7.4.17.
LightCycler 480 Probes Master kit (Roche Diagnostics Cat. No.
04707494001)
15
8.
7.4.18.
ROX reference dye, 25 mM (Invitrogen Cat. No. 12223)
7.4.19.
Hepatitis G virus Armored RNA® (Asuragen Cat. No. 42024)
7.4.20.
Enterovirus, norovirus GI, norovirus GII Armored RNA (Asuragen
custom order giving >1010 genomic copies at a defined concentration)
QUALITY ASSURANCE
This section describes the minimum quality assurance (QA) requirements. Laboratories are
encouraged to institute additional QC practices that go beyond these minimum criteria to
meet their needs.
8.1.
QUALITY ASSURANCE PLAN
All laboratories analyzing samples with this method must adhere to defined quality
assurance procedures that ensure analytical data which are scientifically valid and
which demonstrate acceptable precision and specificity. Each laboratory must have a
written Quality Assurance Plan that addresses the following:
8.1.1.
Laboratory organization and responsibility – This section must include the
following: 1) a list that identifies the laboratory QA manager(s) and key
individuals who are responsible for ensuring the production of valid
measurements and the routine assessment of QC data, 2) specify who is
responsible for internal audits and reviews of the implementation of the
QA plan and its requirements, and 3) include a chart showing the
laboratory organization and line authority.
8.1.2.
Personnel – This section must list each analyst’s academic background
and experience, describe how each analyst is trained to perform the
method, and describe how training is documented.
8.1.3.
Facilities – This section must describe the arrangement and size of
laboratories, workflow patterns to minimize cross contamination, air
system; laboratory reagent water system, and waste disposal system [see
reference (36)].
8.1.4.
Field sampling procedures – This section must describe the laboratory
chain-of-custody procedures, including the sample identification and
information recording system; and describe how field samples are
collected and transported, including transportation time and temperature.
8.1.5.
Laboratory sample handling procedures – This section must describe
sample-holding times and temperature during analyses and the procedures
for maintaining the integrity of the samples, i.e., logging and tracking of
samples from receipt through analyses and disposal.
8.1.6.
Equipment – This section must describe the specifications, calibration
procedures, preventive maintenance, and how quality control records are
maintained for each item used during the performance of the method. All
calibrations must be traceable to national standards, when they are
available.
16
8.2.
8.1.7.
Supplies – This section must describe the specifications, storage
conditions, and how catalog and lot numbers are documented for
chemicals, reagents, and media.
8.1.8.
Laboratory practices – This section must describe the preparation of
reagent-grade water, glassware washing and preparation procedures, and
sterilization procedures. It should also describe the workflow
requirements among laboratories to prevent cross contamination,
especially for molecular procedures. The work flow and other
recommended requirements are described in detail in Sen et al. (36).
8.1.9.
Analytical procedures – This section must reference this method and
identify available laboratory SOPs.
8.1.10.
Quality control checks – This section must describe all laboratory
procedures that are implemented to ensure the quality of each analyst’s
data.
8.1.11.
Data reduction, verification, and reporting – This section must describe
any procedures for converting raw to final data, identify procedures for
ensuring the accuracy of data transcription and calculations, and describe
the laboratory’s procedures for reporting all data to EPA.
8.1.12.
Corrective actions – This section must describe how the laboratory will
respond to PE and QC failures and from failures of its own internal QC
procedures, identify the person(s) responsible for taking corrective action,
and describe how the effectiveness of the actions will be documented.
8.1.13.
Record keeping – This section must describe how records are maintained
(e.g., hard copy, electronic or LIMS, etc.), how long records are kept, and
where records are stored.
LABORATORY PERSONNEL
8.2.1.
Principal Analyst/Supervisor – Laboratories must have a principal analyst
who may also serve as a supervisor if an additional analyst(s) is to be
involved. The principal analyst/supervisor oversees or performs the entire
analyses and carries out QC performance checks to evaluate the quality of
work performed by analysts and technicians. This person must be an
experienced microbiologist with at least a B.A./B.S. degree in
microbiology or a closely related field. The person must also have a
minimum of three years continuous bench experience in cell culture
propagation, processing and analysis of virus samples, and in performing
PCR, and at least six months of experience in performing RT-qPCR. This
analyst must have analyzed a PE sample set and results must fall within
acceptance limits. The principal analyst must also demonstrate acceptable
performance during any on-site performance audits.
8.2.2.
Analyst – The analyst performs at the bench level under the supervision of
a principal analyst and can be involved in all aspects of analysis, including
preparation of sampling equipment, filter extraction, sample processing,
cell culture, virus assay, qPCR, and data handling. The analyst must have
17
two years of college lecture and laboratory course work in microbiology
or a closely related field. The analyst must have at least six months bench
experience in cell culture, animal virus analyses and PCR, including three
months experience in filter extraction of virus samples and sample
processing. Six months of additional bench experience in the above areas
may be substituted for the two years of college. Each analyst must have
analyzed a PE sample set and results must fall within acceptance limits.
The analyst must also demonstrate acceptable performance during any an
on-site audits. Should laboratories choose to use teams of analysts who
specialize in performing the culture or molecular portions of this method,
analysts only need to meet the educational requirement of the portion they
perform. Laboratories using analyst teams must ensure that the PE
samples are analyzed by the appropriate team member.
8.3.
8.2.3.
Technician – The technician extracts filters, processes samples, and
performs qPCR under the supervision of an analyst, but does not perform
cell culture work, virus detection, or enumeration. The technician must
have at least three months experience in filter extraction, processing of
virus samples to participate in the cultural portion of this method and three
months of experience with PCR to participate in the molecular portion of
the method.
8.2.4.
Samplers – The sampler collects water samples and ships them to the
analytical laboratory. The sampler must be familiar with the sample
collection process and have at least training by means of a video or written
instructions demonstrating proper sampling technique. Laboratories are
responsible for ensuring that samplers have adequate training.
LABORATORY PERFORMANCE
8.3.1.
Laboratories using this method must evaluate the ability of analysts to
perform the method using known quality control (QC) and unknown
performance evaluation (PE) samples as defined in Section 8.3.1.2 and
8.3.1.3. EPA may also require laboratories to be approved.
8.3.1.1.
Laboratory approval – Laboratories must have a Quality
Assurance Plan, adequately trained staff, proper equipment,
and at least one approved analyst to be approved.
8.3.1.2.
Initial analyst approval/initial demonstration of capability –
Each analyst must demonstrate the ability to perform the
method using QC and PE samples as part of an initial approval
process. New analysts must initially use QC samples to gain
method proficiency followed by the analysis of a PE sample
set.
8.3.1.3.
Ongoing analyst approval/ongoing demonstration of capability
– Each analyst must analyze one QC sample set for every
analysis batch (see section 3) and one PE sample set per month
following initial approval to remain approved. A QC sample
set is comprised of a negative and a positive QC sample (see
18
section 8.4). A PE sample set is comprised of two PE samples
that may or may not contain virus at various levels (section
8.5). For initial and ongoing approval, analysts must meet the
method performance characteristics defined in section 14 or in
any additional guidance from EPA. Any analyst who does not
meet the initial or ongoing demonstration of capability must
not process samples until the cause of the failure has been
identified and corrected.
8.4.
QC SAMPLE SET
A QC sample set consists of a negative and a positive QC sample. QC stocks (item
7.1.2) with a titer of 200 MPN/mL should be prepared by the analytical laboratories
and stored in aliquots containing about 1.1 mL at -70°C. QC sample results must
meet the method performance characteristics defined in section 14.
8.4.1.
Note: It is difficult to obtain an accurate pH on pure water. To
compensate, a buffering agent, such as HEPES (Sigma Aldrich Cat. No.
H4034), may be added to the water at a concentration up to 0.01M.
8.4.2.
Note: QC samples should use the filter type (e.g., 1MDS or NanoCeram)
that will be used for collecting samples. If both filter types are being used,
the QC samples should be performed using the filter type that is most
frequently analyzed by the analyst.
8.4.3.
Negative QC Sample/Equipment blanks
8.4.4.
8.4.3.1.
Place 10 L of reagent grade water in a dispensing pressure
vessel or polypropylene container (e.g., Cole Parmer Product
No. EW-06317-53). Adjust the pH to 6.5-7.5 with 0.12 M HCl
or 0.1 M NaOH, as necessary.
8.4.3.2.
Place a magnetic stir bar into the vessel or container and stir for
10 minutes at a speed sufficient to create a vortex.
8.4.3.3.
Pass the water through a sterile standard filter apparatus
(Section 6.1) containing a sterile electropositive filter using a
flow rate of approximately 10 L/minute. Note: to meet
ongoing QC requirements standard filter apparatuses that have
been sterilized after use in the field must be used.
8.4.3.4.
Process and analyze the filter using the Filter Elution (section
10), Organic Flocculation (section 11), Total Culturable Virus
Assay (section 12) and Enterovirus and Norovirus Molecular
(section 13) procedures.
Positive QC Sample
8.4.4.1.
Place 10 L of reagent grade water in a dispensing pressure
vessel or polypropylene container (e.g., Cole Parmer Product
No. EW-06317-53). Adjust the pH to 6.5-7.5 with 0.12 M HCl
or 0.1 M NaOH, as necessary.
19
8.4.5.
8.5.
8.6.
8.4.4.2.
Add 1.0 mL of a QC sample (see item 7.1.2) to the water.
8.4.4.3.
Place a magnet into the vessel or container and stir for 10
minutes at a speed sufficient to create a vortex.
8.4.4.4.
Pass the water through a sterile standard apparatus containing a
sterile electropositive filter using a flow rate of approximately
10 L/minute.
8.4.4.5.
Process and analyze the filter using the Elution (step 10),
Organic Flocculation (step 11), Total Culturable Virus Assay
(step 12) and Enterovirus and Norovirus Molecular (step 13)
procedures.
QC sample stocks (item 7.1.2) are also to be used for the Positive Assay
Control (see item 7.3.3 and step 12.1.2.3.2).
PE SAMPLES
8.5.1.
PE samples will be sent to analysts in a randomized fashion and may
contain no, low, medium, or high levels of Sabin poliovirus type 3 on the
filter type (e.g., 1MDS or NanoCeram) used for sampling.
8.5.2.
Process and analyze the PE filter using the Elution (section 10), Organic
Flocculation (section 11), Total Culturable Virus Assay (section 12) and
Enterovirus and Norovirus Molecular (section 13) procedures and
according to any additional requirements supplied with the samples.
8.5.3.
PE sample results should meet the method performance characteristics
defined in section 14.
MATRIX SPIKE
Matrix spike should be run for every sample location initially and then after every
20th sample from the same location. Matrix spikes duplicates are performed by
collecting two samples at the sampling location as described in section 9, except
that the sampling volume of the second sample is reduced by 10 L. The last 10L is
collected in a 10 L cubitainer (item 6.2.3), shipped back to the laboratory, seeded
with 1 mL of the matrix spike (item 7.1.3), passed through the duplicate filter, and
analyzed by the method procedures (steps 10 through 12.2.11). The results of the
analysis of the matrix spike must meet the performance measures in section 14.
8.7.
RECORD MAINTENANCE
Laboratories shall maintain all records related to data quality. This shall include a
record of the analyst name, date, and results of all QA controls performed, records
of equipment calibration and maintenance, and reagent and material catalog and lot
numbers used for all analytical procedures.
9.
SAMPLE COLLECTION, PRESERVATION, AND STORAGE
9.1.
SAMPLE COLLECTION
9.1.1.
Filter sampling apparatus sterilization
20
9.1.1.1.
Before each use, analytical laboratories must wash, and then
sterilize the intake and cartridge housing modules, any
necessary injector modules, and pumps as described in section
15.2.4.
9.1.1.2.
Cover the apparatus module ends and the injector port(s) with
sterile aluminum foil.
9.1.1.3.
Place the injector module and tubing into a sterile bag or
wrapping in such a way that they may be removed without
contaminating them.
9.1.1.4.
Ship the filter sampling apparatus components to the
individuals who will be collecting water samples.
9.1.2.
Preparation for sample collection
9.1.3.
Note: Individuals collecting water samples for virus analysis must wear
surgical gloves and avoid conditions that can contaminate a sample with
virus. Gloves should be changed after touching human skin or handling
components that may be contaminated (e.g., water taps, other
environmental surfaces). Care must be taken to ensure that cartridge
filters are properly seated in the housings. Housings with properly seated
filters must not leak. Filters should be checked for proper seating upon
opening the housing at the analytical laboratory by examining the gaskets
for depressions that do not extend beyond the edge of the filter.]
9.1.3.1.
Purge the water tap to be sampled before connecting the filter
apparatus. Continue purging for 2-3 minutes or until any
debris that has settled in the line has cleared.
9.1.3.2.
Remove the foil from the backflow regulator. Loosen the
swivel female insert slightly to allow it to turn freely and
connect the backflow regulator to the tap. Retighten the swivel
female insert. Disconnect the cartridge housing module at the
quick connect, if connected, and cover the open end with sterile
foil.
9.1.3.3.
Remove the foil from the ends of the discharge module and
connect it to the regulator module. Place the end of the
regulator module or the tubing connected to the outlet of the
regulator module into a 1 L plastic bottle.
9.1.3.4.
Slowly turn on the tap and adjust the globe valve until the flow
meter/totalizer reads 10 L/min. If the tap is incapable of
reaching this flow rate, adjust the valve to achieve the
maximum flow rate. Slower flow rates will result in longer
sampling times.
9.1.3.5.
Flush the apparatus assembly with at least 75 L of the water to
be sampled. While the system is being flushed, measure the
21
chlorine residual, pH, temperature, and the turbidity of the
water collecting in and overflowing from the 1 L plastic bottle.
9.1.4.
9.1.5.
9.1.3.6.
Record the pH, temperature, and turbidity values onto a sample
data sheet.
9.1.3.7.
Turn off the water at the tap.
Injector module adjustment (Note: If a NanoCeram filter is being used
and if the water pH is 9.0 or less and if it does not contain a disinfectant,
skip to section 9.1.5. If disinfected waters above pH 8.0 are being used
with a 1MDS cartridge filter, substitute a double injector module for the
single injector module in the following steps. With 1MDS filters under
these conditions, use a second metering pump connected to the second
connection on the double injector module to add 0.12 M HCl at a rate,
which brings the pH of the water exiting the discharge module to 6.5 to
7.5.)
9.1.4.1.
If the sample contains a disinfectant, turn off the water at the
tap and disconnect the discharge module from the regulator
module.
9.1.4.2.
Remove the foil from the ends of an injector module and
connect the module to the quick connect of the regulator
module.
9.1.4.3.
Turn on the metering pump. Set the pump to deliver 2.4 or
6±0.2 mL/minute for flow rates of 4 or 10 L/minute,
respectively (see Table 2). Measure the flow exiting the
injector module for several minutes to ensure that the flow rate
is correct. Measure the chlorine residual and if present, readjust the flow rate until no residual is present. Re-mark the
setting, if necessary. Turn off the metering pump.
Virus collection
9.1.5.1.
If connected, remove the discharge module. Remove the foil
from the cartridge housing module and connect it to the end of
the regulator module, or if used, the injector module. Connect
the discharge module to the outlet of the cartridge housing
module.
9.1.5.2.
If the water sample has turbidity greater than 75 NTU, remove
the foil from each end of the prefilter module and connect the
prefilter module between the regulator module or, if used, the
injector module and the cartridge housing module.
9.1.5.3.
Record the unique sample number, location, date, time of day
and initial totalizer reading onto a sample data sheet (section
17.1).
9.1.5.4.
If an injector module is being used, turn on the metering pump.
22
9.1.5.5.
9.2.
With the filter housing placed in an upright position, slowly
open the water tap until it is completely open (Note: If the
cartridge housing has a vent button, press it while opening the
tap to expel air from the housing. When the air is totally
expelled from the housing, release the button, and open the
sample tap completely. If the housing does not have a vent
button, allow the housing to fill with water before completely
opening the tap).
9.1.5.5.1.
After the tap is opened completely, check the flow
rate and readjust to the recommended rate from
Table 2, if necessary.
9.1.5.5.2.
Check and readjust the metering pump rate, if
necessary.
9.1.5.6.
Using the totalizer readings, pass a volume of water through
the apparatus that equals the volume specified in Table 2.
9.1.5.7.
Turn off the flow of water at the sample tap at the end of the
sampling period and record the date, time of day, and totalizer
reading onto a sample data sheet (see section 17.1). Although
the totalizer reading may be affected by the addition of
thiosulfate, the effect is insignificant and may be ignored.
9.1.5.8.
Loosen the swivel female insert on the regulator module and
disconnect the backflow regulator from the tap. Disconnect the
cartridge housing module and the prefilter housing module, if
used from the other modules.
9.1.5.9.
Turn the filter housing(s) upside down and allow excess water
to flow out. Turn the housing(s) upright and cover the quick
connects on each end of the modules with sterile aluminum
foil.
SHIPMENT OF SAMPLES
9.2.1.
Pack the cartridge housing module(s) into an insulated shipping box.
9.2.2.
Add 6-8 small ice packs (prefrozen at -20°C) or double bagged ice cubes
around the cartridge housings to keep the sample cool in transit (the
number of ice packs or bags may have to be adjusted based upon
experience to ensure that the samples remain cold, but not frozen).
9.2.3.
9.2.2.1.
Add an iButton (or other temperature recording devise) to a
location in the shipping box where it will not come in direct
contact with the ice packs or bags.
9.2.2.2.
The temperature during shipment must be in the range of 110°C.
Drain and add the regulator and injector modules used.
23
9.3.
9.2.4.
Place the sample data sheet (protected with a closable plastic bag) in with
the sample.
9.2.5.
Drain and then cover the ends of the discharge module with foil. The
discharge module may remain at the sampling site, if samples will be
taken on a routine basis. If not, pack the module into the shipping box.
9.2.6.
Close the insulated portion of the shipping box and tape to prevent any
leakage of water. Close and label.
9.2.7.
If the shipping box cannot be directly transported to the laboratory for
virus analysis by close of business on the day collected or by the next
morning, ship it to the laboratory by overnight courier.
LABORATORY HOLDING TIME AND TEMPERATURE
9.3.1.
9.3.2.
Record the date of arrival and the arrival condition on the sample data
sheet packed with the sample. Print out the transit temperature reading
from the iButton.
9.3.1.1.
Attach the readout of the iButton or other temperaturerecording device for recording the temperature during shipment
to the sample data sheet.
9.3.1.2.
Warning: The cartridge filters must arrive from the utility or
other sampling site in a refrigerated, but not frozen, condition.
The temperature during shipment must be in the range of 110°C.
9.3.1.3.
Brief transient temperatures outside the acceptable range
associated with the initial packing and closing of the shipping
box and its opening at the analytical laboratory may be
ignored.
Filters must be refrigerated immediately upon arrival. Ideally, viruses
should be eluted from filters within 24 h of the start of the sample
collection, but all filters must be eluted within 72 h of the start of the
sample collection.
10. FILTER ELUTION PROCEDURE
10.1. ELUTION EQUIPMENT SETUP
10.1.1.
Attach sections of braided tubing to the inlet and outlet ports of the
cartridge housing containing the cartridge filter (see Figure 4). Note: If a
prefilter or more than one electropositive filter was used to collect a
sample, each filter must be eluted and analyzed separately using the
procedures below.
10.1.2.
Place the sterile end of the tubing connected to the outlet of the cartridge
housing into a sterile 2 L glass or polypropylene beaker.
24
10.1.3.
Connect the free end of the tubing from the inlet port of the cartridge
housing to the outlet port of a sterile pressure vessel and connect the inlet
port of the pressure vessel to a positive air pressure source.
10.2. ELUTION
10.2.1.
10.2.2.
First elution
10.2.1.1.
Elute NanoCeram or 1MDS filters with 500 mL or 1,000 mL of
buffered 1.5% beef extract, pH 9.0 (item 7.2, prewarmed to
room temperature), respectively, by opening the cartridge
housing and adding a sufficient amount of beef extract to cover
the filter. Close the housing and pour the remaining beef
extract into the pressure container. An acceptable alternative to
the use of a pressure vessel is to use a peristaltic pump and
sterile tubing to push the remaining beef extract through the
filter.
10.2.1.2.
Replace the top of the pressure vessel and close its vent/relief
valve.
10.2.1.3.
Open the vent/relief valve (if present) on the cartridge housing
and slowly apply sufficient pressure to fill the housing with
beef extract. Close the vent/relief valve (if present) as soon as
the buffered beef extract solution begins to flow from it.
Carefully observe housings without vents to ensure that all
trapped air has been purged.
10.2.1.4.
Turn off the pressure and allow the solution to contact the filter
for 1 minute. Wipe up spilled liquid with disinfectant-soaked
sponge.
10.2.1.5.
Increase the pressure to force the buffered beef extract solution
through the filter(s). The solution should pass through the
filter slowly to maximize the elution contact period. When air
enters the line from the pressure vessel, elevate and invert the
filter housing to permit complete evacuation of the solution
from the filters. Note: Slow passage of the solution also
minimizes foaming, which may inactivate some viruses; the
addition of a few drops of sterile antifoam to minimize foaming
is optional.
10.2.1.6.
Turn off the pressure at the source and open the vent/relief
valve on the pressure vessel.
Second elution
10.2.2.1.
For the NanoCeram filter, repeat sections 10.2.1.1 to 10.2.1.6
using an additional 500 mL of buffered 1.5% beef extract,
except increase the contact time in section 10.2.1.4 to 15
minutes. For a 1MDS filter, place the buffered beef extract
25
from the 2 L beaker back into the cartridge housing and
pressure vessel and repeat sections 10.2.1.2 to 10.2.1.6).
10.2.2.2.
Turn off the pressure at the source and open the vent/relief
valve on the pressure vessel. Combine the two 500 mL
portions from the elution of NanoCeram filters. Record the
total volume on the Virus Data Sheet (17.2). Thoroughly mix
the eluate and proceed to the Organic Flocculation
Concentration Procedure immediately.
11. ORGANIC FLOCCULATION CONCENTRATION PROCEDURE
11.1. ORGANIC FLOCCULATION
11.1.1.
Place a sterile stir bar into the beaker containing the buffered beef extract
eluate from the cartridge filter. Place the beaker onto a magnetic stirrer,
and stir at a speed sufficient to develop a vortex. Minimize foaming
(which may inactivate viruses) throughout the procedure by not stirring or
mixing faster than necessary to develop a vortex.
11.1.2.
Sterilize the electrode of a combination-type pH electrode as described in
section 15.2.4. Calibrate the pH meter at pH 4 and 7.
11.1.3.
Insert the sterile pH electrode into the beef extract eluate. Add 1.2 M HCl
to the eluate dropwise while moving the tip of the pipette in a circular
motion away from the vortex to facilitate mixing. Continue adding 1.2 M
HCl until the pH reaches 3.5 ± 0.1.
11.1.4.
While continuing to monitor the pH, slowly stir the eluate for 30 minutes
at room temperature. A precipitate will form during the 30 minutes
stirring period. If pH falls below 3.4, add 1 M NaOH to bring it back to
3.5 ± 0.1. Exposure to a pH below 3.4 may result in virus inactivation.
11.1.5.
Remove the electrode from the beaker, and pour the contents of the beaker
into a centrifuge bottle. To prevent the transfer of the stir bar into a
centrifuge bottle, hold another stir bar or magnet against the bottom of the
beaker while decanting the contents. The beef extract suspension will
usually have to be divided into several centrifuge bottles.
11.1.6.
Cap the bottle and centrifuge the precipitated beef extract suspension at
2,500 x g for 15 minutes at 4°C.
11.1.7.
Carefully pour off or aspirate the supernatant so as to not disturb the
pellet, including any loose floc on top of the pellet, and then discard the
supernatant.
11.2. RECONCENTRATED ELUATE
11.2.1.
Place a stir bar into the centrifuge bottle that contains the precipitate.
11.2.2.
Add 30 mL of 0.15 M sodium phosphate, pH 9.0. Note: A smaller
volume (down to 15 mL) may be used, if the analytical laboratory’s PE
sample sets meet the performance requirements of Section 14.
26
11.2.3.
11.2.4.
Place the bottle onto a magnetic stirrer, and stir slowly until the precipitate
has dissolved completely. Continue stirring for at least 10 minutes before
proceeding to step 11.2.3.4. When the centrifugation is performed in more
than one bottle, dissolve the precipitates in a total of 30 mL and combine
into one bottle before proceeding to the next step. Significant virus loss
can occur if the precipitates are not dissolved completely. Precipitates that
prove to be difficult to dissolve can be treated using any of the following
techniques:
11.2.3.1.
Break up the precipitate with a sterile spatula before or during
the stirring procedure.
11.2.3.2.
Use a pipette repeatedly to draw the solution up and down
during the stirring.
11.2.3.3.
Shake the precipitate at 160 rpm for 20 minutes on an orbital
shaker in place of stirring.
11.2.3.4.
If the above procedures take longer than 20 minutes to dissolve
the precipitate or if experience with the water matrix shows
that precipitates are always difficult to manage, either slowly
adjust the pH to 7.0 - 7.5 with 1.2 M HCl or resuspend the
precipitate initially in 0.15 M sodium phosphate, pH 7.0 - 7.5.
Use one of the above techniques to dissolve the precipitate and
then slowly re-adjust the pH to 9.0 with 1 M NaOH. Mix for
10 minutes at room temperature before proceeding.
11.2.3.5.
Check the pH and readjust to 9.0 with 1 M NaOH, as
necessary. Remove the stir bar.
11.2.3.6.
Centrifuge the dissolved precipitate at 4,000 x g for 10 minutes
at 4°C. The centrifugation speed may be increased to 10,000 x
g for 10 minutes at 4°C to facilitate the filtration step below.
Record the centrifugation speed on the virus data sheet (section
17.2).
11.2.3.7.
Remove and collect the supernatant and discard the pellet.
Adjust the pH of the supernatant to 7.0-7.5 slowly with 1.2 M
HCl.
11.2.3.8.
Pretreat a sterilizing filter (item 6.3.7) or, for samples that are
difficult to filter, a sterilizing filter stack (item 6.3.9) with 1015 mL of 1.5% beef extract (item 7.2.4). Load the supernatant
into a 50 mL syringe and force it through the filter. If the
sterilizing filter or filter stack begins to clog badly, empty the
loaded syringe into the bottle containing the unfiltered
supernatant, fill the syringe with air, and inject air into filter to
force any residual sample from it. Continue the filtration
procedure with another filter.
Calculation of assay volumes and preparation of subsamples
27
11.2.4.1.
Record the reconcentrated eluate volume (designated the Final
Concentrated Sample Volume for cultural assays; FCSV) on
the virus data sheet (section 17.2).
11.2.4.2.
Calculate the Assay Sample Volume (S) for source and
finished water samples using equation 1. D (the Volume of
Original Water Sample Assayed) is the amount of
reconcentrated eluate that must be assayed by Total Culturable
Virus Assay (section 12) or processed for the Enterovirus and
Norovirus Molecular Assay (section 12.2.11). This amount is
100 L for source water or 500 L for finished or ground waters.
TSV is the Total Sample Volume from the Virus Data Sheet.
The Assay Sample Volume is the volume of the
reconcentrated eluate that represents 100 L of source water or
500 L of finished or ground waters. Record the S and D onto
the Virus Data Sheet (section 17.2).
S
D
 FCSV
TSV
Equation 1
11.2.4.3.
For example, if 1,800 L of a groundwater sample is passed
through the NanoCeram filter and subsequently concentrated to
30 mL, then TSV equals 1,800 L, D equals 500 L, FCSV
equals 30 mL, and S = (500 L/1,800 L) x 30 mL = 8.33 mL.
11.2.4.4.
Prepare three subsamples of the reconcentrated eluate. Prepare
subsamples 1 and 2 with a volume equal to 1.2 times the Assay
Sample Volume. Prepare subsample 3 with the remaining
volume. Label subsamples 1-3 with appropriate sampling
information for identification.
11.2.4.5.
11.2.4.4.1.
Hold subsample 1 at 4°C for use with the Total
Cultural Virus Assay if it can be assayed within
24 hours, otherwise freeze at -70°C.
11.2.4.4.2.
Hold subsample 2 at 4°C and analyze using the
Molecular Assay within 24 hours. Note: Freeze
thawing leads to norovirus losses.
11.2.4.4.3.
Freeze subsample 3 at -70°C for backup and
archival purposes.
Determine the Inoculum Volume for the Total Culturable
Virus Assay (section 12) by dividing the Assay Sample
Volume by 10. Record the Inoculum Volume onto the Virus
Data Sheet (section 17.2).
11.2.4.5.1.
For ease of inoculation, a sufficient quantity of
0.15 M Na2HPO4, pH 7.0 - 7.5, may be added to
the Inoculum Volume to give a more usable
working Final Inoculation Volume (e.g., 1.0
mL). If this option is used, then record the Final
28
Inoculation Volume onto the Virus Data Sheet
(section 17.2) and substitute the term Final
Inoculation Volume for each use of the
Inoculum Volume, except where indicated.
11.2.4.5.2.
11.2.4.6.
For example, if an Inoculum Volume of 0.72 mL
is to be placed onto each of 10 vessels, then for
10.5 vessels (with the extra 0.5 added to account
for sample loss on the surface of the vessel used
for the preparation), 7.56 mL of subsample will be
needed (10.5 x 0.72 mL). The addition of 2.94
mL of 0.15 M Na2HPO4, pH 7.0-7.5 (10.5 x (10.72 mL) to the 7.56 mL of subsample results in a
mixture that contains the required Inoculum
Volume per 1.0 mL.
Calculate the Assay Sample Volume for QC (section 8.4)
samples by multiplying FCSV by 0.3. Calculate the Inoculum
Volume by dividing the Assay Sample Volume by 10. Divide
the Final Concentrated Sample Volume from QC samples
into three subsamples and handle as described in step 11.2.4.4.
12. TOTAL CULTURABLE VIRUS ASSAY
12.1. QUANTAL ASSAY
12.1.1.
12.1.2.
Preparation of cell culture test vessels
12.1.1.1.
Using 10 cell culture test vessels for every water sample to be
tested, code each vessel with the water sample number,
subsample number, analyst initials, and date using an indelible
marker.
12.1.1.2.
Return the cell culture test vessels to a 36.5 ± 1°C incubator
and hold at that temperature until the cell monolayer is to be
inoculated.
12.1.1.3.
Decant and discard the medium from cell culture test vessels.
12.1.1.4.
Wash the test vessels with a balanced salt solution (e.g., item
7.3.2.1) or maintenance medium without serum using a wash
volume of at least 0.06 mL/cm2 of surface area. Rock the wash
medium over the surface of each monolayer several times and
then decant and discard the wash medium. Do not disturb the
cell monolayer during the wash procedure.
Inoculation of test samples
12.1.2.1.
Rapidly thaw subsample 1, if frozen, and inoculate an amount
equal to the Inoculum Volume or the Final Inoculation
Volume (see step 11.2.4.5) onto each of 10 cell culture test
vessels.
29
12.1.2.2.
12.1.2.1.1.
Note: Samples should be thawed in a 37°C water
bath or under warm running water at about 37°C
with shaking. Samples should be removed from
the warm water as soon as the last ice crystal melt.
12.1.2.1.2.
Note: The number of cell culture replicates was
cut from the 20 required by the ICR standard
method (19) to 10 to reduce labor costs. This
reduction of replicates results in wider 95%
confidence limits (c. 20-40%) and reduces the
maximum virus titer that can be assayed without
dilutions by about 25%. Laboratories assaying
samples that are known to have more than about
20 MPN/100 L may assay subsample 1 using
more than 10 replicates or by using dilutions as
described in section 12.1.2.2.
12.1.2.1.3.
Note: The analysis of a second subsample is not
required for this method. Subsample 2 was used
in the ICR method (19) to account for
cytotoxicity. Samples with cytotoxicity should be
assayed using dilutions as described in section
12.1.2.2
12.1.2.1.4.
Warning! Use at least a different pipetting tip or
device for each set of samples to be inoculated.
For QC samples and for water samples known or suspected of
having virus concentrations greater than 20 MPN/100 L,
prepare five- and twenty five-fold dilutions of subsample 1 (or
subsample 3, see section 12.1.2.1.3).
12.1.2.2.1.
Prepare a 1:5 dilution by adding a volume of
subsample 1 equal to 0.1334 times the Assay
Sample Volume (amount "a") to a volume of 0.15
M sodium phosphate (pH 7.0-7.5) equal to 0.5336
times the Assay Sample Volume (amount "b").
Mix thoroughly.
12.1.2.2.2.
Prepare a 1:25 dilution by adding amount "a" of
the 1:5 diluted sample to amount "b" of 0.15 M
sodium phosphate (pH 7.0-7.5).
12.1.2.2.3.
Inoculate 10 cell cultures each with undiluted
subsample 1, subsample 1 diluted 1:5, and
subsample 1 diluted 1:25, respectively, with an
amount equal to the Inoculum Volume.
12.1.2.2.4.
Freeze the remaining portions of the 1:25 dilution
at -70°C until the sample results are known.
Thaw and perform additional 5-fold dilutions
30
using the dilution format above if all replicates of
the undiluted to 1:25-fold dilutions develop CPE.
12.1.2.3.
Cell culture controls
12.1.2.3.1.
Negative Assay Control – Inoculate three or
more BGM cultures with a volume of sodium
phosphate, pH 7.0 - 7.5, equal to the Inoculum
Volume as a negative control. Run a Negative
Assay Control with every group of subsamples
inoculated onto cell cultures. If any Negative
Assay Control develops CPE, all subsequent
assays should be halted until the cause of the
positive result is determined.
12.1.2.3.2.
Positive Assay Control – Inoculate three or more
BGM cultures with the Positive Assay Control
(see item 7.3.3). Run a positive control with
every group of subsamples inoculated onto cell
cultures. This control will provide a measure for
continued sensitivity of the cell cultures to virus
infection. If any Positive Assay Control fails to
develop CPE, all subsequent assays should be
halted until the cause of the negative result is
determined.
12.1.2.4.
Record the date of inoculation on the Virus Data Sheet
(section 17.2).
12.1.2.5.
Rock the inoculated cell culture test vessels gently to achieve
uniform distribution of inoculum over the surface of the cell
monolayers. Place the cell culture test vessels on a mechanical
rocking platform set at 1-5 oscillations per minute at room
temperature.
12.1.2.6.
Continue incubating the inoculated cell cultures for 80 - 120
minutes at room temperature to permit viruses to adsorb onto
and infect cells. Note: If a rocking platform is not available,
the vessels may be placed upon a level laboratory surface, but
the vessels should be rocked every 15-20 minutes during the
adsorption period to prevent cell death in the middle of the
vessels from dehydration.
12.1.2.7.
Add liquid maintenance medium (see Item 3.7) and incubate at
36.5 ± 1°C.
12.1.2.7.1.
Warm the maintenance medium to 36.5 ± 1°C
before placing it onto cell monolayers.
12.1.2.7.2.
Add the medium to the side of the cell culture
vessel opposite the cell monolayer.
31
12.1.3.
12.1.4.
12.1.5.
12.1.2.7.3.
Warning! Never touch the pipetting device to the
inside rim of the cell culture test vessels during
medium addition. This step represents the most
likely place where cross contamination of cultures
can occur. Cross contamination will result in
invalid MPN values and can cause false positive
results. Laboratories must ensure that analysts
take great precaution in performing this step.
12.1.2.7.4.
If CPE has not started to develop, the cultures
may be re-fed with fresh maintenance medium
after 4-7 days.
CPE development
12.1.3.1.
Examine each culture microscopically for the appearance of
CPE daily for the first three days and then every couple of days
for a total of 14 days.
12.1.3.2.
Freeze cultures at -70°C when more than 75% of the
monolayer has developed CPE. Freeze all remaining cultures,
including controls, after 14 days.
Perform a second passage for confirmation. Confirmation passages may
be performed in small vessels or multiwell trays, however, it may be
necessary to distribute the inoculum into several vessels or wells to insure
that the inoculum volume is less than or equal to 0.04 mL/cm2 of surface
area.
12.1.4.1.
Thaw all the cultures, including the negative and positive assay
controls, to confirm the results of the previous passage.
Refreeze at least 2 mL of the medium from each vessel at 70°C for optional analysis by molecular methods (see step
13.3.2).
12.1.4.2.
Filter at least 10% of the medium from each vessel that was
positive for CPE through separate 0.22 μm sterilizing filters
(item 6.3.8). If the medium is difficult to filter, it can be
centrifuged at 1,500 to 18,000 x g prior to filtration.
12.1.4.3.
Prepare fresh cell culture test vessels as described in step
12.1.1. Inoculate the fresh cultures with the thawed medium
from all negative samples (step 12.1.4.1) and the filtered
medium from step 12.1.4.2 using an inoculation volume that
represents 10% of the medium from the first passage.
12.1.4.4.
Repeat sections 12.1.2.4 to 12.1.3.1.
Score cultures that developed CPE in both the first and second passages as
confirmed positives.
12.1.5.1.
Perform a third passage as described in Step 12.1.3.2 through
12.1.4.4 on any cell culture vessels that were negative during
32
the first passage and positive in the second passage and with
the negative assay controls. Other vessels that were either
negative or positive in both the first and second passages do
not need to be carried through the third passage.
12.1.5.2.
Score cultures that develop CPE in both the second and third
passages as confirmed positives.
12.2. VIRUS QUANTITATION
12.2.1.
Record the total number of confirmed and not confirmed positive and
negative cultures for each subsample onto a Total Culturable Virus Data
Sheet (section 17.3).
12.2.2.
Transfer the number of cultures inoculated and the number of confirmed
positive cultures from the Total Culturable Virus Data Sheet for each
sample to the Quantitation of Total Culturable Virus Data Sheet
(section 17.4).
12.2.3.
Calculate the MPN/mL value (MmL) and the upper (CLumL) and lower
(CLlmL) 95% confidence limits/mL using the number of confirmed
positive cultures from step 12.2.2 and the MPN computer program
supplied by U.S. EPA (www.epa.gov/microbes). If dilutions are required,
calculate the MPN/mL value and 95% confidence limits using the
confirmed values from all dilutions, including dilutions with all positive or
negative samples.
12.2.3.1.
Change the MPN program default settings as shown in Table 3.
12.2.3.2.
Note that the Inoculum Volume rather than the Inoculation
Volume (see step 11.2.4.5) must be used.
12.2.3.3.
The MPN program will run on Windows XP and later versions.
It has been re-designed for calculation of both standard
bacterial and viral MPN values. All entries are saved in a
default database and can be viewed to check for data entry
errors using the View History selection under the Tools menu.
Each program run can also be saved into Word, Excel, or text
files for transfer to lab notebooks or to Laboratory Information
Management Systems.
12.2.4.
Place the values obtained onto the Quantitation of Total Culturable
Virus Data Sheet (section 17.4).
12.2.5.
Calculate the MPN per L value (ML) of the original water sample using
equation 2 where S equals the Assay Sample Volume and D equals the
Volume of Original Water Sample Assayed (the values for S and D can
be found on the Virus Data Sheet). Record the value of ML onto the
Virus Data Sheet (section 17.2). For MmL values of 0, calculate the
sample detection limit rather than the ML value by dividing 1 by D.
Report as equal to or less than the calculated detection limit. For samples
where more than one cartridge filter or a prefilter was used, place the total
33
MPN/L and Confidence Limits/L (from steps 12.2.7 and 12.2.9) values for
all filters on the Virus Data Sheet and place the individual totals for each
filter under “Other Comments.”
ML =
M mL S
D
Equation 2
12.2.6.
For example, if the sample described in the example in step 11.2.4.2,
which has an Inoculum Volume equal to 0.833 mL, had 4 positive
replicates, the MPN/mL value is 0.61 with 95% Confidence Limits of 0.12
to 1.31. The MPN per L value then equals (0.61 MPN/mL x 8.33 mL)/500
L = 0.0102.
12.2.7.
Calculate the lower 95% confidence limit per L value (CLL) for each
water sample using equation 3 where CLlmL is the lower 95% confidence
limit per milliliter from the Quantitation of Total Culturable Virus Data
Sheet. Record the limit per L values on the Virus Data Sheet.
CLL 
CLlmL S
D
Equation 3
12.2.8.
Continuing with the example from step 12.2.5, CLL = (0.12 MPN/mL x
8.33 mL)/500 L = 0.002.
12.2.9.
Calculate the upper 95% confidence limit per L value (CLU) using
equation 4 where CLumL is the upper 95% confidence limit per milliliter
from the Quantitation of Total Culturable Virus Data Sheet. Record
the limit per L values on the Virus Data Sheet.
CLU 
CLumL S
D
Equation 4
12.2.10. Continuing with the example from step 12.2.5, CLU = (1.31 MPN/mL x
8.33 mL)/500 L = 0.0218.
12.2.11. Calculate the total MPN value and the total 95% confidence limit values
for each QC samples by multiplying the values per milliliter by S and
dividing by 0.3.
13. ENTEROVIRUS AND NOROVIRUS MOLECULAR ASSAY
The molecular assay uses RT-qPCR based upon standard curves to provide a quantitative
estimate of enterovirus and norovirus genomic copies per liter in environmental and
drinking waters. Only microliter volumes can be analyzed by RT-qPCR, so the procedure
includes additional concentration of any viruses present in the sample beyond that required
for culture. Surface and ground waters may contain substances that interfere with RTqPCR, so the assay uses an RNA extraction kit to reduce inhibition and a control to identify
samples that are inhibitory to RT-qPCR. The assay uses primers and probes from the
scientific literature (Table 4). The primer/probe sets are designed to detect many
enteroviruses and noroviruses. Standard curves are prepared from Armored RNA®
reagents that contain the target sequence for the primer/probe sets. Armored RNA was
chosen for standard curves because it is difficult to obtain high titered norovirus stocks.
34
13.1. PRELIMINARY PROCEDURES
13.1.1.
Purchase the primers and probes shown in Table 4.
13.1.2.
Prepare 100 µM stock solutions of each oligonucleotide primer (probes
should come from the manufacturer at 100 μM solutions).
13.1.2.1.
Centrifuge the vials containing primers in a microcentrifuge for
30 seconds.
13.1.2.2.
Dissolve the primers in a volume of molecular or PCR grade
water (e.g., Roche Cat. No. 03 315 932 001) that equals the
number of nanomoles of primer shipped (as identified on the
specification sheet from the manufacturer) times 10 (in µl; e.g.,
if a primer contains 51.0 nmol; resuspend in 510 µl).
13.1.2.3.
Measure the absorbance at 260 nm. Calculate the total
absorbance by multiplying the absorbance value by the volume
used to resuspend the primer (and by any dilution required to
obtain the absorbance reading). Confirm that the reading is the
same as the total absorbance value recorded on the
manufacturer’s specification sheet. Note: Probes should be
quality checked using this same procedure.
13.1.2.4.
Aliquot the stock solutions into small quantities to avoid
multiple freeze thaw cycles and store at -20ºC.
13.1.3.
Prepare 10 µM primer and probe working solutions by diluting the stock
solutions 1:10 (or by a dilution that compensates for any differences
between the absorbance reading obtained in Section 13.1.2.3 and the
manufacturer’s specification sheet) in molecular or PCR grade water.
Aliquot stocks and store at -20ºC.
13.1.4.
Note: Preparation of primers and probes must be performed in a clean
room or other location to minimize the possibility of false positive
reactions. A clean room or location is one in which molecular and
microbiological procedures are not performed.
13.2. TERTIARY CONCENTRATION OF SUBSAMPLE 2
13.2.1.
For each sample to be analyzed, label a Vivaspin 20 unit with the sample
number, analyst’s initials and date, and fill the unit with PBS, 0.2% BSA
(item 7.4.5). Soak at least 2 hours at room temperature or overnight at
4ºC.
13.2.2.
Discard the PBS, 0.2% BSA from the Vivaspin 20 unit and add an amount
of the appropriate subsample 2 (see section11.2.4.4.2) equal to the Assay
Sample Volume (S).
13.2.3.
Centrifuge at 3,000 x g with swinging buckets until the sample has been
concentrated down to about 50 μL.
13.2.4.
Add 1 mL of sterile 0.15 M sodium phosphate, pH 7-7.5, and repeat step
13.2.3. Repeat one additional time.
35
13.2.5.
Transfer the concentrate to a 1.5 ml microcentrifuge tube. Measure the
volume and add 0.15 M sodium phosphate, pH 7-7.5 to bring the total
volume to 0.4 mL.
13.2.6.
Immediately proceed to step 13.3 or hold at 4°C for no more than 72
hours. Note: Freeze/thawing leads to norovirus losses.
13.3. NUCLEIC ACID ISOLATION
13.3.1.
Preliminary procedures
13.3.1.1.
Add 310 μL of Buffer AVE (item 7.4.8) to a vial of carrier
RNA (from item 7.4.7) to give a final concentration of 1
μg/μL.
13.3.1.2.
Aliquot and store at -20°C. Prepare a sufficient number of
aliquots so that each aliquot does not have to be frozen and
thawed more than three times.
13.3.1.3.
Add carrier RNA to Buffer AVL (item 7.4.7; note: do not use
the Buffer AL that comes with the Qiagen kit) to give a
concentration of 0.027 μg/μL by adding, per sample, 5.6 μL of
carrier RNA (from step 13.3.1.2) to 200 μL of Buffer AVL
(e.g., 5.6 μL carrier RNA × number of samples + 200 μL
Buffer AVL × number of samples).
13.3.2.
For each sample to be processed, label a 1.5 mL microcentrifuge tube and
add 200 μL of the appropriate sample from section 13.2.5 (or from section
12.1.4.1 for confirmation of culture positive samples) and vortex briefly to
mix. Freeze any remaining tertiary concentrate at -70°C.
13.3.3.
Add 200 μL of Buffer AVL with carrier RNA from step 13.3.1.3. Vortex
for 15 seconds.
13.3.4.
Incubate at 56°C for 10 minutes.
13.3.5.
Centrifuge at >5,000 rpm for 5 seconds in a microcentrifuge.
13.3.6.
Add 200 μL of ethanol. Vortex for 15 seconds and then centrifuge for
about 5 seconds.
13.3.7.
Add the mixture to a QIAamp Mini Spin column, but avoid wetting the
rim of the tube. Close the cap.
13.3.8.
Centrifuge at 6,000 x g for 1 minute. Check to determine if the sample
has completely passed through the column. If it has not, centrifuge again
for 1 minute at 10,000-20,000 x g or for longer times until the sample has
completely passed through the column.
13.3.9.
Place the Mini Spin column into a clean 2 mL collection tube (item 7.4.9)
and discard the collection tube containing the filtrate.
13.3.10. Add 500 μL of Buffer AW1 without touching the tube rim.
13.3.11. Centrifuge at 6,000 x g for 1 minute. Again, transfer the column to a clean
collection tube and discard the tube containing the filtrate.
36
13.3.12. Add 500 μL of Buffer AW2 without touching the tube rim.
13.3.13. Centrifuge at 20,000 x g for 3 minutes. Again, transfer the column to a
clean collection tube and discard the tube containing the filtrate.
13.3.14. Centrifuge at 20,000 x g for 1 minute.
13.3.15. Add 40 units of RNasin to a clean microcentrifuge tube. Transfer the
column from the collection tube to the 1.5 mL microcentrifuge tube and
discard the collection tube. Alternatively, RNasin can be added to an
amount of Buffer AE sufficient for the number of samples to be eluted at a
concentration of 400 units/mL in place of adding it to the microcentrifuge
tubes.
13.3.16. Add 50 μL of Buffer AE to the column. Incubate at room temperature for
1 minute and then centrifuge for 1 minute at 6,000 x g.
13.3.17. Repeat step 13.3.16. Remove and discard the column.
13.3.18. Proceed immediately to step 13.4 or prepare aliquots and store the RNA at
-70°C until it can be assayed.
13.4. REVERSE TRANSCRIPTION (RT)
13.4.1.
13.4.2.
Preliminary procedures (to be performed in a clean room)
13.4.1.1.
Label PCR plates or tubes with appropriate sample numbers.
13.4.1.2.
Prepare RT master mix 1 and 2 using the guide in Table 5. The
amounts shown for the volume per master mix can be scaled up
or down according to the number of samples that need to be
analyzed.
13.4.1.3.
Vortex the master mixes after the addition of all ingredients.
13.4.1.4.
Aliquot 16 μL of RT master mix 1 to the labeled PCR tubes or plate wells.
13.4.2.1.
Run every environmental water, QC, and PE sample in
triplicate by adding 6.7 μL of the appropriate sample to each of
the tubes or plate wells labeled for that sample. Note: It is not
necessary to prepare 1:5 and 1:25 dilutions of the QC samples
as done for the culture assay (section 12.1.2.2). See Figure 4
for a schematic of the RT-qPCR process.
13.4.2.2.
Add 6.7 μL of molecular grade water to one or more tubes or
plate wells as no template controls (NTC). Include at least one
NTC for the replicates associated with every fourth water
sample run on a plate; NTC controls must be distributed
throughout the plate. Note: If any NTC control is positive, the
cause of the false positive value should be investigated. After
fixing the cause of the problem, all samples on the plate must
be rerun.
37
13.4.2.3.
13.5.
Close the tubes or seal the plates and heat at 99°C for 4
minutes, followed by quenching on ice or a hold temperature of
4°C.
13.4.3.
Add 17.3 μL of RT mix 2 to each tube or well.
13.4.4.
Centrifuge at >5,000 rpm for 10 seconds in a microcentrifuge at 4°C.
13.4.5.
Run at 25°C for 15 minutes, 42°C for 60 minutes, 99°C for 5 minutes, and
then hold at 4°C for up to 2 hours until PCR is performed. Note: Thermal
cyclers from a number of different manufacturers can be used for this and
the following real-time quantitative PCR step. Analysts must follow the
manufacturers’ instructions for set-up, runs, and analysis for the
instrument used.
13.4.6.
13.4.7.
Proceed immediately to step 13.5 or store reverse transcribed samples at 70°C until they can be processed.
REAL-TIME QUANTITATIVE PCR (qPCR)
13.5.1.
Preliminary procedures
13.5.1.1.
13.5.2.
Label PCR plates or tubes with appropriate sample numbers.
13.5.1.1.1.
Note: each sample will require 15 plate wells or
tubes (i.e., 3 RT replicates times 5 PCR assays;
see Figure 4).
13.5.1.1.2.
To save reagents, the HGV inhibition control
assay may be run before all other qPCR assays
(see section 13.5.8).
13.5.1.2.
Prepare PCR master mixes using the guides in Tables 6-10.
The amounts shown for the volume per master mix can be
scaled up or down according to the number of samples that
need to be analyzed.
13.5.1.3.
Vortex the master mix after the addition of all ingredients.
13.5.1.4.
13.5.1.5.
Dispense 14 μL of the appropriate mix to the labeled plates or
tubes.
Add 6 μL of the appropriate sample to each tube (see Figure 4).
13.5.3. Run on a thermal cycler with a setting of 1 cycle at 95°C for 10 minutes
followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute.
13.5.4.
Analyze the results of each run to calculate the Genome Copy (GC)
numbers of unknown samples according to the instructions of the
manufacturer of the thermal cycler used based upon the standard curve
samples described in step 13.5.9.
38
13.5.5.
Record the GC values on the Molecular Virus Results Data Sheet
(section 17.6) of each replicate and mean and standard deviation of the
three replicates for each sample. Include non-detects (zeros) in the
calculation of the mean
13.5.6.
Calculate the Genomic Copies per L (GCL) for each sample using
equation 5 and the mean GC value from step 13.5.4, where DF equals the
reciprocal of any dilution performed to compensate for inhibition (see step
13.5.8; e.g., 5 and 25, or 1 for undiluted samples) and D equals the
Volume of Original Water Sample Assayed (see step11.2.4.2).
GC L 
GC  199  DF
D
Equation 5
For example, if the PCR assay from the sample example described in step
11.2.4.2 detects 15 genomic copies in a 1:5 dilution, then the number of
genomic copies per 100 L is 100 x ((15 x 199 x 5)/500 L) =2985. Note:
199 is the total dilution factor for the volume reductions that occur in steps
13.2 through 13.5.
13.5.6.1.
Record the GCL value on the Molecular Virus Results Data
13.5.6.2.
For water samples with a mean value of zero, report the
Genomic Copies per L as less than or equal to the detection
limit (i.e., ≤1/D).
13.5.7. Calculate the genomic copies of QC samples by multiplying the mean
genomic copy value by 199 and dividing by 0.3.
13.5.8. Inhibition Control
13.5.8.1.
Hepatitis G Armored RNA is used as an inhibition control
rather than specific enterovirus and norovirus to reduce the
possibility of cross-contamination that could occur between
seeded and unseeded samples with the actual virus groups
being tested.
13.5.8.2.
Before running any water samples, run ten replicate RT and
hepatitis G qPCR assays to determine the mean hepatitis G Cq
value and standard deviation.
13.5.8.2.1. Process a volume of FCSV from at least two
negative QC controls (step 11.2.4.6) equal to the
Assay Sample Volume (S) using steps 13.2 to
13.3. Use 6.7 μL of the RNA from one of these
samples; hereafter designated “negative FCSV”
for five of the replicates and 6.7 μL from the other
sample for the other five replicates.
13.5.8.2.2. Calculate the mean quantitation cycle (Cq) value
and standard deviations. Record the results on the
Molecular Virus Results Data Sheet (section
39
17.6). The mean value should be between 25 and
32 Cq units. The standard deviation of the mean
should be <0.3 units.
13.5.8.2.3. Note: If the mean value is not between 25 and 32
Cq units, readjust the amount of Hepatitis G
Armored RNA added to RT Master Mix 1 (see
Table 5) and repeat step 13.5.8.2 until the value is
within the acceptable range.
13.5.8.3.
Compare the hepatitis G Cq values obtained with all samples
against the value calculated in step 13.5.8.2. If the value in the
unknown samples is more than 1 Cq value higher than that
calculated in step 13.5.8.2 dilute the unknown sample 1:5 and
1:25 in molecular grade (nuclease-free) water.
13.5.8.4.
Re-run the sample along with the 1:5 and 1:25 dilution.
13.5.8.5.
Because some samples will demonstrate some level of
inhibition, the hepatitis G assay can be run with all samples
prior to doing the other qPCR assays as stated in step
13.5.1.1.2. All other assays can then be run without dilution
for samples giving no inhibition and with the appropriate
dilution for all other samples.
13.5.8.6.
Calculate the sample concentration using the highest dilution
for which the hepatitis G Cq values are within 1 unit of the
value calculated in step 13.5.8.2. If the inhibition control fails
again and the sample Cq value is lower than 38, re-run the
sample at higher five-fold dilutions. If any sample run at the
higher dilution fail the inhibition control again or if any
unknown samples are below the detection limit (e.g., Cq values
of 45 or higher), list the sample as a potential false negative
sample on the Molecular Virus Results Data Sheet (section
17.6)
13.5.9. Preparation of standard curves. Note: Analysts must run new standard
curves (a single standard curve for each Armored RNA standard) with
every set of unknown samples run together or use calibrators (see step
13.5.10) with stored standard curves, if the thermal cycler allows standard
curves to be stored.
13.5.9.1.
Dilute enterovirus, norovirus GI and norovirus GII Armored
RNA in negative FCSV to give a concentration of 2.5×106
particles/250 μL (i.e., 1×107/mL) based upon the concentration
of the Armored RNA lots supplied. Note: unless specified
otherwise by EPA, transcribed RNA from plasmids containing
the appropriate viral sequence or poliovirus, norovirus GI, and
norovirus GII stocks may be substituted for the Armored RNA.
40
13.5.9.1.1. Transcribed RNA must be titered using standard
methods, e.g., see reference (32), and genomic
copies per mL substituted for particles per mL
above.
13.5.9.1.2. Virus stocks must be titered using RT-qPCR on
endpoint serial dilutions and ten replicates per
dilution for each stock. The MPN calculator must
then be used to obtain the titer of the stock and the
MPN value substituted for particles per mL
above. If the efficiency of the standard curve is in
the acceptable range (see step 13.5.9.7), genome
copies per mL can be substituted for MPN/mL.
13.5.9.2.
Prepare 6 ten-fold dilutions to give concentrations of 1×106,
1×105, 1×104, 1×103, 1×102, 1×101 and 1 × 100 particles/mL.
Prepare by serially adding 25 μL of Armored RNA to 225 μL
of negative FCSV to create each dilution with vortexing for 515 seconds before proceeding to the next higher dilution.
13.5.9.3.
Run 200 μL of each dilution separately through steps 13.313.5.4 using the volumes described in the steps and only the
specific primers/probe for the Armored RNA standard. This
will result in six standards with 100 to 105 particles per assay.
13.5.9.4.
Calculate the standard curve slope and R2 values for each
standard curve by plotting Cq values against the log of the
concentration for each point or, if available, by using the slope
and R2 values determined by the qPCR instrument.
13.5.9.5.
Calculate the mean of the slopes and standard deviation of the
mean for the three runs and the mean of the R2 values. Note:
An acceptable standard curve will have a mean R2 value >0.97
and a standard deviation of <0.25. Standard deviations of 0.25
or higher represent errors in preparing dilutions or in pipetting.
13.5.9.6.
Calculate the percent amplification efficiency using equation 6.
Record the results on the Molecular Virus Protocol Data
% Efficiency  100  (10 1 slope  1)
Equation 6
13.5.9.7.
For example, the ideal efficiency occurs when the slope equals
-3.32, where the % Efficiency = 100 x (10-1/-3.33 – 1) = 100 x
(2.0-1) = 100. Note: An acceptable standard curve will have an
amplification efficiency of 80-110%.
13.5.9.8.
Standard curves that meet the criteria specified in steps
13.5.9.5 and 13.5.9.6 above must be used to calculate genomic
copies of unknown samples in step 13.5.4.
13.5.10. Preparation of calibrators and standard curves for use with calibrators
41
13.5.10.1. Stored standard curves must be used with calibrators. The
stored standard curves must be based upon the mean of three
runs with each set of standards. New standard curve sets must
be generated as described above every eighth sample batch (see
section 3) or every two months, whichever comes first, or
anytime a calibrator fails twice in a row to meet acceptance
criteria.
13.5.10.2. To prepare calibrators for each virus standard, choose the
dilution from the standard curve that gives the Cq value closest
to, but not greater than 32. Note: calibrators must be run with
all unknown samples if stored standard curves are used, but
may be run even if standard curves are run with each test as an
additional quality check.
13.5.10.3. Prepare the dilution corresponding to the chosen value in
negative FCSV and extract the RNA as described in step 13.3.
Prepare a sufficient number of dilutions to last for the entire
study, taking into consideration that each 200 μL extraction
will yield sufficient material for about 20 runs. Aliquot into
single run batches and store at -70°C.
13.5.10.4. Run a set of at least ten calibrators from each Armored RNA
standard. Calculate the mean Cq value and standard
deviations. Record the mean and standard deviation values as
the Target Value on the Molecular Virus Results Data Sheet
(section 17.6). The standard deviation must be less than 0.25
units.
13.5.10.5. Run all calibrators with every set of unknown samples. Accept
a run if the value of the calibrator for each corresponding PCR
assay falls within 1.0 Cq units of the calibrators’ mean values.
Record the observed value for each target virus as the Actual
Value on the Molecular Virus Results Data Sheet (section
17.6).
13.5.10.6. Reject sample sets run in a PCR assay where the calibrator for
that assay falls outside the acceptance criteria. Repeat the run
once upon failure. If the assay fails again, generate a new
standard curve or take steps to determine the cause of the
failure.
14. METHOD PERFORMANCE
14.1. CULTURABLE ASSAY
14.1.1. This method is subject to a number of biases that reduce its precision and
accuracy. The isoelectric point of the virus particle affects its ability to
bind to, and be eluted from, electropositive filters. The isoelectric point
can vary significantly across virus species and even within members of the
same species. Other capsid and matrix related characteristics and
42
substances could affect virus recovery at various stages of the method.
The passage number of the BGM cell line and the media used to passage
and maintain cells is known to affect the ability of viruses to replicate in
cells. The performance characteristics given below are based upon Sabin
poliovirus type 3 and may not be reflective of other viruses that are
detected by this method. The best performance data for the method comes
from the performance evaluation (PE) samples that were analyzed during
the ICR as described (19). In total, 12 laboratories with 25 ICR-approved
analysts analyzed 828 PE samples, consisting of low (<300 MPN per
filter), medium (300-1,500 MPN per filter) and high (>1,500 MPN per
filter) virus levels. The mean interlaboratory recovery was 56% with a
coefficient of variation (CV) of 92%, a false negative rate of 1.3%, and a
false positive rate of 1.1%. The highest mean recoveries values (71%)
were obtained with the PE samples containing the low virus levels. Table
11 gives the mean recovery and CV value ranges for individual analysts
and intralaboratory variation. Although Method 1615 uses a different
electropositive filter than the ICR study, both filters have been shown to
give the same recoveries in a single (24) and a four laboratory validation
study (unpublished data).
14.1.2. The detection limit of the culture method is 0.01 MPN/L for surface water
and 0.002 MPN/L for groundwater.
14.1.3. Acceptance limits of PE and QC samples is set for this method at a mean
recovery of 35 to 150% with a CV of less than or equal to 70% based upon
the 90% confidence limits of the data from the ICR PE analyses.
14.2. MOLECULAR PROCEDURE
14.2.1. The molecular procedure is subject to the same bias as the culturable
procedure in terms of virus adsorption and recovery from the
electropositive filters and secondary concentration procedures. Additional
bias can occur during tertiary concentration, RNA elution and RT-qPCR.
The method was tested using seven groundwater samples from five
different wells with a range of physicochemical characteristics. In
addition to bias from matrix effects, these tests may have had additional
bias because they were performed as described in section 8.6. The seven
groundwater samples gave a mean recovery of 26% with a recovery range
of 5-60% and a CV of 73%. These same samples were also tested for
norovirus recovery using Murine norovirus and Murine norovirus specific
primers and probe with the Method 1615 protocols. Mean recovery of
Murine norovirus was 35% with a recovery range of 7-63% and a CV of
69%.
14.2.2. As with the culture method, the acceptance criteria for the molecular
procedure is based upon the variation among PE samples seeded with
Sabin poliovirus type 3. The acceptance criteria for the molecular
procedure is a mean recovery of 20 to 150% with a CV of less than or
equal to 75% based upon genomic copies detected by the molecular
43
procedure and the genomic copies seeded onto the PE samples, unless
specified otherwise by EPA for specific applications.
14.2.3. The detection limit of the molecular method based upon the overall
detection limit of the RT-qPCR assay and the volume of water sample
assayed. The detection limit for the poliovirus assay is about 2 and 0.4
genomic copies/L for surface water and groundwater, respectively. The
detection limit can be increased by running more than three RT-qPCR
replicates from each sample.
14.3. PERFORMANCE RECORD
The laboratory should maintain a record of the performance of QC and PE samples
for both the cultural and molecular portions of this method and measure each
individual performance against the ongoing laboratory values and the performance
data given in this section. This should be done by continuously updating the mean
recovery and coefficient of variation values with the addition of each new QC and
PE sample.
15. STERILIZATION AND DISINFECTION
15.1. GENERAL GUIDELINES
15.1.1. Use aseptic techniques for handling test waters, eluates and cell cultures.
15.1.2. Sterilize apparatus and containers that will be exposed to test waters and
all solutions that will be added to test waters unless otherwise indicated.
Thoroughly clean all items before final sterilization using laboratory
standard operating procedures.
15.1.3.
Sterilize all contaminated materials before discarding.
15.1.4. Disinfect all spills and splatters.
15.2. STERILIZATION TECHNIQUES
15.2.1. Solutions
15.2.1.1.
Sterilize all solutions, except those used for cleansing, standard
buffers, HCl, NaOH, and disinfectants by autoclaving them at
121°C for at least 15 minutes.
15.2.1.2.
HCl, NaOH, and disinfectants used are self-sterilizing. When
autoclaving buffered beef extract, use a vessel large enough to
accommodate foaming.
15.2.2. Autoclavable glassware, plasticware, and equipment
15.2.2.1.
General considerations: Add sufficient dH2O to all vessels to
be autoclaved to equal about 1-2% of the vessel’s rated
volume. Water speeds the sterilization process by enhancing
the transfer of heat. Place large vessels on their sides in the
autoclave, if possible, to facilitate the displacement of air in the
vessels by flowing steam.
44
15.2.2.2.
Cover the openings into autoclavable glassware, plasticware,
and equipment loosely with aluminum foil before autoclaving.
Autoclave at 121°C for at least 30 minutes.
15.2.2.3.
Glassware may also be sterilized in a dry heat oven at a
temperature of 170°C for at least 1 hour.
15.2.2.4.
Sterilize stainless steel vessels (dispensing pressure vessel) in
an autoclave at 121°C for at least 30 minutes. Vent-relief
valves on vessels so equipped must be open during autoclaving
and closed immediately when vessels are removed from
autoclave.
15.2.2.5.
Pre-sterilize 1MDS filters and prefilters by wrapping the filters
in Kraft paper and autoclaving at 121°C for 30 minutes.
Warning: Do not autoclave the NanoCeram filters specified in
item 6.1.2.4. These filters are sterilized by the manufacturer
and have housings that cannot be autoclaved.
15.2.3. Sterilize instruments, such as scissors and forceps, by immersing them in
95% ethanol and flaming them between uses.
15.2.4. Sodium hypochlorite sterilization - Sterilize pumps, plasticware (filter
housings) and tubing that cannot withstand autoclaving, and vessels that
are too large for the autoclave with sodium hypochlorite (item 7.1.4).
Ten-inch cartridge prefilters, but not NanoCeram or 1MDS filters, may be
presterilized with sodium hypochlorite as an alternative to autoclaving.
Filter apparatus modules should be disinfected after use by sterilization
and then cleaned according to laboratory standard operating procedures
before final sterilization.
15.2.4.1.
Sterilize the items that cannot be autoclaved by recirculating or
immersing the items in 0.525% sodium hypochlorite for 30
minutes. pH electrodes should be sterilized with 0.525%
hypochlorite for at least 5 minutes.
15.2.4.2.
Drain the hypochlorite from the objects being sterilized and
rinse in sterile water.
15.2.4.3.
Dechlorinate by recirculating or immersing the items in a
solution containing 50 mL of 1M sodium thiosulfate per liter of
sterile dH2O. . Ensure that the sodium hypochlorite (step
15.2.4.1) and sodium thiosulfate (step 15.2.4.3) solutions come
in full contact with all surfaces when performing this
procedure.
15.2.4.4.
Cover the apparatus module ends and the injector port(s) with
sterile aluminum foil.
15.2.4.5.
Place the injector module and tubing into a sterile bag or
wrapping in such a way that they may be removed without
contaminating them.
45
15.2.5. Contaminated materials
15.2.5.1.
Autoclave contaminated materials for at least 30 minutes at
121°C. Be sure that steam can enter contaminated materials
freely.
15.2.5.2.
Many commercial disinfectants do not adequately kill enteric
viruses. To ensure thorough disinfection, disinfect spills and
other contamination on surfaces with either a solution of 0.5%
iodine (item 7.2.2) or 0.525% sodium hypochlorite. The iodine
solution has the advantage of drying more rapidly on surfaces
than sodium hypochlorite, but may stain some surfaces.
46
16.
TABLES AND FIGURES
Table 1. Viruses Detected by EPA Method 1615
Detected by TCVAa
Detected by qPCR
Human enterovirus A
Some serotypes
Yes
Human enterovirus B
Most serotypes
Yes
Human enterovirus C
Some serotypes
Yes
Human enterovirus D
Some serotypes
Yes
Norovirus genogroup I and II
No
Many genotypes
Mammalian orthoreovirus
Yes
No
Virus Genus or Species
a
TCVA – Total Cultural Virus Assay (see section 12)
Table 2. Specified and Recommended Sample Volumes
Water Type
Flow Ratea
(L/min)
Sampling duration
(hours)
Sample Volume
(liters)b,c
Sewage effluent
10
0.2
120d
Surface
10
0.6
360e
Finished/Groundwater
10
3.0
1,800f
Finished/Groundwater
4
16±2
≤4,320g
a
Poliovirus retention is independent of flow rates between 4 and 20 L/minute for
NanoCeram filters, but a constant flow rate, such as describe here, should be used for any
single study (24). EPA may specify alternative flow rates for specific studies.
b
Consistent sample volumes should be used for any single study. EPA may specify
alternative sample volumes for specific studies.
c
Turbidity and other factors may affect the volume collected during any sampling event.
The sample duration must be increased to meet the specified or recommended volume
during these situations. As an alternative, two cartridge filter modules may be used to
obtain the specified volume.
d
This is a recommended value for final sewage effluents. There is no recommended
volume for raw sewage.
e
The minimum specified volume is 300 L for surface waters.
f
The minimum specified volume is 1,500 L for treated tap or untreated groundwater.
g
For convenience, samples may be collected by starting the sampling at the end of a work
day and stopping it in the morning of the next day.
47
Table 3. MPN Program Default Settings for MPN Calculator
Item
Default Setting
Data Entry Mode
Keyboard
Dilution type
Standard 5-Fold Serial
Approximation type
Cornish & Fisher Limits
Confidence Level
95%
Number of Dilutions
1 (or 3 or actual number of dilutions)
Number of Tubes per dilution
10
Inoculum volume (mL)
The Inoculum Volume used (see section
11.2.4.5)
Table 4. Primers and TaqMan® Probes for Virus Detection by RT-qPCR
Virus
group
Primer pairsa,b
TaqMan Probec,d
De Leon et al. (17)
CCTCCGGCCCCTGAATG
Enterovirus
CGGAACCGACTACTTTGGGTGTCCGT
ACCGGATGGCCAATCCAA
Norovirus
GIA
GCCATGTTCCGITGGATG
Norovirus
GIB
CGCTGGATGCGNTTCCAT
Norovirus
GII
ATGTTCAGRTGGATGAGRTTCTCWGA
Hepatitis G
Reference
Monpoeho et
al.(30)
TGTGGACAGGAGATCGCAATCTC
Jothikumar et al.
(23)
TGGACAGGAGAYCGCRATCT
Butot et al. (12)
AGCACGTGGGAGGGGATCG
Butot et al. (12)
AGGTCCCTCTGGCGCTTGTGGCGAG
Schlueter et al.
(35)
TCCTTAGACGCCATCATCAT
CCTTAGACGCCATCATCATTTAC
TCGACGCCATCTTCATTCACA
CGGCCAAAAGGTGGTGGATG
CGACGAGCCTGACGTCGGG
a
EPA may specify additional or alternative primer sets for specific applications. Degenerate
bases in primers and probes are as follows: N equals a mixture of all four nucleotides; R
equals A + G; Y equals T + C; and W equals A + T.
b
The top primer in each set is the forward primer and the bottom primer is the reverse primer.
All primer and probe sequences are 5’ to 3’.
c
Probes are labeled on the 5’-end with 6-FAM and with TAMRA on the 3’-end.
d
Primers and probes are designated in Tables 6-10 by the first three letters of the virus name
followed by F, R, or P for forward, reverse, and probe. GIA, GIB, or GII are also added to the
norovirus designations.
48
Table 5. RT Master Mix 1 and 2
Ingredient
Volume per
Reaction (μL)a
Final Concentration
Volume per
Master Mix (μL)b
0.8
10 ng/μL (c. 5.6 μM)
77.6
RT Mix 1
Random Primer
Hepatitis Gc
PCR grade water
Total
1
97
14.2
1377.4
16
1552
RT Mix 2
4
10 mM tris, pH 8.3,
50 mM KCL
388
25 mM MgCl2
4.8
3 mM
465.6
10 mM dNTPs
3.2
0.8 mM
310.4
100 mM DTT
4
10 mM
388
RNase Inhibitord
1
0.5 units/μL
97
SuperScript II RT
0.3
1.6 units/μL
29.1
Total
17.3
10X PCR Buffer II
1678.1
a
The volumes given are for 40 μL RT assays.
b
The volumes shown should be multiplied by the number of reactions to be performed plus
an additional 0.5-1 reaction to account for losses during transfer of the master mix to tubes
or plates. The amounts shown are sufficient for a 96 well PCR plate.
c
Hepatitis G Armored RNA is supplied as an untitered stock. The amount to use should be
determined for each lot as described in section 13.5.8.2.
d
The amount shown is for a stock concentration of 20 unit/μL. The manufacturer’s stock
concentration varies between 20 and 40 units/μL. Amounts need to be adjusted for stocks
with higher concentrations.
49
Table 6. PCR Master Mix for Enterovirus Assay
Ingredient
Volume per
Reaction (μL)a
Final Concentration
Volume per Master
Mix (μL)b
2X LightCycler 480
Probes Master Mixc
10
Proprietary
970
ROX reference dyed
0.4
0.5 mM
38.8
PCR grade water
1
97
EntF
0.6
300 nM
58.2
EntR
1.8
900 nM
174.6
EntP
0.2
100 nM
19.4
Total
14
1358
a
The volumes given are for using 6 μL of cDNA from Step 13.4.7 in a qPCR assay using a total
qPCR volume of 20 μL.
b
The volumes shown should be multiplied by the number of reactions to be performed plus an
additional 0.5-1 reaction to account for losses during transfer of the master mix to tubes or
plates. The amounts shown are sufficient for a 96 well PCR plate.
c
10X PCR Buffer II (2 μL/reaction), 25 mM MgCl2 (5 μL/reaction), and AmpliTaq Gold (0.2
μL/reaction) can be substituted for the LightCycler 480 Probe Master mix.
d
This reagent is necessary for use with Applied Biosystems and similar instruments. It should
be substituted with PCR grade water for use with the LightCycler and similar instruments.
Table 7. PCR Master Mix for Norovirus GIA Assay
Ingredient
Volume per
Reaction (μL)a
Final Concentration
Volume per Master
Mix (μL)
2X LightCycler 480
Probes Master Mix
10
Proprietary
970
ROX reference dye
0.4
0.5 mM
38.8
PCR grade water
1.4
135.8
NorGIAF
1
500 nM
97
NorGIAR
1
500 nM
97
NorGIAP
0.2
100 nM
19.4
Total
14
a
1358
See table 6 for legends.
50
Table 8. PCR Master Mix for Norovirus GIB Assay
Ingredient
Volume per
Reaction (μL)a
Final Concentration
Volume per Master
Mix (μL)
2X LightCycler 480
Probes Master Mix
10
Proprietary
970
ROX reference dye
0.4
0.5 mM
38.8
PCR grade water
0.3
29.1
NorGIBF
1
500 nM
97
NorGIBR
1.8
900 nM
174.6
NorGIBP
0.5
250 nM
48.5
Total
14
1358
a See table 6 for legends.
Table 9. PCR Master Mix for Norovirus GII Assay
Ingredient
Volume per
Reaction (μL)a
Final Concentration
Volume per Master
Mix (μL)
2X LightCycler 480
Probes Master Mix
10
Proprietary
970
ROX reference dye
0.4
0.5 mM
38.8
PCR grade water
0.3
29.1
NorGIF
1
500 nM
97
NorGIR
1.8
900 nM
174.6
NorGIP
0.5
250 nM
48.5
Total
14
a
1358
51
Table 10. PCR Master Mix for Hepatitis G Assay
Ingredient
Volume per
Reaction (μL)a
Final Concentration
Volume per Master
Mix (μL)
2X LightCycler 480
Probes Master Mix
10
Proprietary
970
ROX reference dye
0.4
0.5 mM
38.8
PCR grade water
1.4
135.8
HepF
1
500 nM
97
HepR
1
500 nM
97
HepP
0.2
100 nM
19.4
Total
14
a
1358
Table 11. Mean Recovery and Coefficient of Variation Range
Variation Type
Mean Recovery Range
CV Range
Individual Analysts
36-98
34-157
Intralaboratory
36-85
58-131
52
Figure 1. Uninfected BGM Cells
Figure 2. Early CPE from Poliovirus
53
Figure 3. Sample Filtration Apparatus
Figure 4. Elution of an Electropositive Filter with Beef Extract
54
Figure 5. RT-qPCR Schematic
Each sample is reverse transcribed in triplicate (RT1, RT2, RT3) using 6.7 μL of extracted
sample RNA for each RT assay in a 40 μL assay volume. Five qPCR assays (EV PCR, NoV
GIA PCR, NoV GIB PCR, NoV GII PCR, and HGV PCR) are run from each of the triplicate RT
reactions using 6 μL of cDNA for each qPCR assay.
55
17. DATA SHEETS
17.1. SAMPLE DATA SHEET
Sample Data Sheet
Sample Number/ID
Utility Name
Site Address
City, State
Sampler’s Namea
Water Type
Location
Surface
waters
Treated
Surface or
Groundwaters
Treatment
Distribution
Plant/Pumping system
Station
Start of Sampling Event
Untreated
Groundwater
Other (specify in
comments section
Other (specify in comments
section)
End of Sampling Event
Date
Time
Totalizer Reading (L)
Flow Rate (L/minute)
Total Sample Volume (L)
Water Parameter Readings
Water Temperature
pH
Turbidity (NTU)
Free Chlorine (mg/L)
Quality Controls
Flow meter model and serial number:
Totalizer model and serial number:
Date of last flow meter/totalizer calibration:
Metering pump model and serial number
Temperature meter model and serial number:
pH meter model and serial number
Turbidity meter model and serial number
Chlorine test meter model and serial number
Metering pump flow rate
Yes
QC check performed
Comments:
a
If any other individuals assist the sampler, include their name in the comments section and add
the initials of the person who performed measurements after the recorded value.
56
17.2. VIRUS DATA SHEET
VIRUS DATA SHEET
Sample Number/ID:
Sample Date:
Sample Arrival Date:
Hold Time Met (Y/N)
Analytical Laboratory Name and ID:
Analytical Laboratory Address:
City:
State:
ZIP:
Analyst Name (Please print or type):
Sample Batch Number:
Date Eluted:
Time:
Eluate Volume Recovered:
L
Date Concentrated:
Time:
Centrifugation Speed (step 11.2.3.6):
xg
Final Concentrated Sample Volume (FCSV):
mL
Volume Of Original Water Sample Assayed (D)b:
L
Assay Sample Volume (S):
mL
Inoculum Volume:
mL
Final Inoculation Volume (If Used):
mL
Dates Assayed By CPE:
1st Passage
2nd Passage
3rd Passage
(If necessary)
Subsample 1a:
95% Confidence Limits/L
MPN/Lc:
Lower:
Upper:
Comments:
Did a heavy floc form during the organic flocculation step? Yes___ No___
Was the floc difficult to dissolve? Yes___ No___
Other comments:
Analyst Signature:
a
Use a separate Virus Data Sheet if it is necessary to assay an additional subsample
e.g., 100 L of surface water or 500 L of finished or ground waters
c
Value calculated from the Quantitation of Total Culturable Virus form as described in the Virus
Quantitation section.
b
57
17.3. TOTAL CULTURABLE VIRUS DATA SHEET
TOTAL CULTURABLE VIRUS DATA SHEET
Sample Number:
Incubator Model and Serial Number:
Passage
Sample
Type
Confirmeda
Total Number of Replicates
Inoculated
Without CPE
With CPE
Neg. Cont.
Pos. Cont.
1st
Undiluted
1:5 Dil.
1:25 Dil.
1:125 Dil.
Neg. Cont.
Pos. Cont.
2ndb
Undiluted
1:5 Dil.
1:25 Dil.
1:125 Dil.
Neg. Cont.
Pos. Cont.
3rdc
Undiluted
1:5 Dil.
1:25 Dil.
1:125 Dil.
a
Place a check (√) next to the negative controls and dilutions that were confirmed.
A portion of medium from each 1st passage vessel, including negative controls, must be
passaged again for confirmation. The terms "Undiluted," "1:5 Dilution" and "1:25 Dilution"
under the 2nd and 3rd Passage headings refer to the original sample dilutions for the 1st passage.
c
Samples that were negative on the first passage and positive on the 2nd passage must be
passaged a third time for confirmation. If a third passage is required, negative controls must be
passaged again.
b
58
17.4. QUANTITATION OF TOTAL CULTURABLE VIRUS DATA SHEET
QUANTITATION OF TOTAL CULTURABLE VIRUS DATA SHEET
SAMPLE NUMBER:
Sample
Number
Replicates
Inoculated
Number
with CPEa
b
MPN/mL
95% Confidence
Limits/mL
Lower
Upper
Undiluted
1:5 Dilution
1:25 Dilution
1:125 Dilution
a
The number of flasks with confirmed CPE from the second passage (or third passage, if
necessary).
b
The MPN/mL and 95% Confidence Limit values must be obtained using the computer
program supplied by the U.S. EPA.
59
17.5. MOLECULAR VIRUS PROTOCOL DATA SHEET
MOLECULAR VIRUS PROTOCOL DATA SHEET
Sample Number:
Analytical Laboratory Name/ID:
City:
State:
ZIP:
Subsample Number:
Sample Batch Number:
Tertiary Concentration
Date:
Time:
Initials:a
Concentrator Cat. No/Lot No.:
b
Assay Sample Volume Concentrated:
mL
Final Tertiary Concentrated Volume:
RNA Extraction
μL
Date:
Time:
Initials:
RNA Extraction Kit Cat. No./Lot No.:
Amount Used For RNA Extraction:
μL
RNA Extract Final Volume:
μL
Reverse Transcription (RT) Step
Master Mixes Prepared
Date:
Time:
Initials:
RNA Extract Volume Used For RT:
RT Samples Run:
μL
Date:
Time:
Initials:
Date:
Time:
Initials:
c
Thermal Cycler Used:
PCR Step
Master Mixes Prepared:
Volume Of RT Used For PCR:
PCR Samples Run
μL
Date:
Time:
Initials:
c
Thermal Cycler/Real Time Instrument Used:
Standard Curves
Enterovirus
Lot #d
Eff.:e
R2
SDf
Norovirus GI
Lot #
Eff.
R2
SD
Eff.
2
SD
Norovirus GII
Lot #
R
a
Record the initials of the analyst at the time this procedure is performed.
The Assay Sample Volume should be the same as recorded on the Virus Data Sheet.
c
Record the make and model of thermal cycler.
d
Assign a new lot number to each standard curve run.
e
Percent efficiency
f
Record the largest standard deviation among the different concentrations of the standard curve lot.
b
60
17.6. MOLECULAR VIRUS RESULTS DATA SHEET
MOLECULAR VIRUS RESULTS DATA SHEET
Sample Number:
Analytical Laboratory Name/ID:
City:
State:
ZIP:
RT-qPCR Results:
All No Template Controls Negative:
Yes
Noa
Sample Type
Target Value (Sd) b
Actual Valuea,c
Inhibitor Control
Enterovirus Calibrator
Norovirus GI Calibrator
Norovirus GII Calibrator
Run Number:d
If Required, Dilution Used In Calibration Of Sample Concentration:
Replicatee
1
2
3
Mean (SD)b
Genomic Copy Values
Genomic Copies per L:f
a
If any no template controls are positive or the inhibition control or calibrator falls outside
specification limits, the samples must be re-run; however, each run should be recorded on this
data sheet.
b
Record the mean and the standard deviation values for the sample type. For water samples,
include non-detects (zeros) in the calculation of the mean.
c
Record the actual Cq value obtained for the sample type
d
A serial record identification of samples that have to be re-run.
e
If more than three replicates are used, record the data from the additional replicates onto
another Molecular Virus Results Data Sheet.
f
Calculate the Genomic Copies per L using Equation 5. For water samples with a mean value of
zero, report the Genomic Copies per L as less than or equal to the detection limit.
61
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